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There has been a long controversy about what defines a species and how to delimitate them which resulted in the existence of more than two dozen different species concepts. Recent research on so-called "cryptic species" heated up this debate as some scientists argue that these cryptic species are only a result of incompatible species concepts. While this may be true, we should keep in mind that all concepts are nothing more than human constructs and that the phenomenon of high phenotypic similarity despite reproductive isolation is real. To investigate and understand this phenomenon it is important to classify and name cryptic species as it allows to communicate them with other fields of science that use Linnaean binomials. To provide a common framework for the description of cryptic species, we propose a possible protocol of how to formally name and describe these taxa in practice. The most important point of this protocol is to explain which species concept was used to delimitate the cryptic taxon. As a model, we present the case of the allegedly widespread Caribbean intertidal mite Thalassozetes barbara, which in fact consists of seven phenotypically very similar but genetically distinct species. All species are island or short-range endemics with poor dispersal abilities that have evolved in geographic isolation. Stabilizing selection caused by the extreme conditions of the intertidal environment is suggested to be responsible for the morphological stasis of this cryptic species complex.
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Molecular Phylogenetics and Evolution 163 (2021) 107240
Available online 29 June 2021
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A taxonomist‘s nightmare Cryptic diversity in Caribbean intertidal
arthropods (Arachnida, Acari, Oribatida)
Tobias Pngstl
, Andrea Lienhard
, Julia Baumann
, Stephan Koblmüller
Institute of Biology, University of Graz, Universitaetsplatz 2, 8010 Graz, Austria
Institute of Applied Production Sciences, FH Joanneum GesmbH, Eckstrasse 7a, 8020 Graz, Austria
Stabilizing selection
Species concept
There has been a long controversy about what denes a species and how to delimitate them which resulted in the
existence of more than two dozen different species concepts. Recent research on so-called cryptic species
heated up this debate as some scientists argue that these cryptic species are only a result of incompatible species
concepts. While this may be true, we should keep in mind that all concepts are nothing more than human
constructs and that the phenomenon of high phenotypic similarity despite reproductive isolation is real. To
investigate and understand this phenomenon it is important to classify and name cryptic species as it allows to
communicate them with other elds of science that use Linnaean binomials. To provide a common framework for
the description of cryptic species, we propose a possible protocol of how to formally name and describe these
taxa in practice. The most important point of this protocol is to explain which species concept was used to
delimitate the cryptic taxon. As a model, we present the case of the allegedly widespread Caribbean intertidal
mite Thalassozetes barbara, which in fact consists of seven phenotypically very similar but genetically distinct
species. All species are island or short-range endemics with poor dispersal abilities that have evolved in
geographic isolation. Stabilizing selection caused by the extreme conditions of the intertidal environment is
suggested to be responsible for the morphological stasis of this cryptic species complex.
1. Introduction
Species are one of the fundamental units of biology making organ-
isms comparable in various biological aspects (e.g. Mayr, 1982). In this
sense, species are nothing more than a human construct allowing bi-
ologists to classify and compare organisms. For that purpose, it is vital to
dene exactly what a ‘speciesis, but different groups of biologists
advocate different denitions (e.g. Harrison, 1998) and so more than
two dozen of sometimes incompatible species concepts exist (de
Queiroz, 2007). This resulted in seemingly endless debates between
advocates of the different concepts whereas de Queiroz (2007) proposed
a unied species concept to end this debate. But his concept, which
denes a species as a separately evolving metapopulation lineage, has
remained widely neglected. The most commonly used concept is still the
biological species concept, that denes a species as a group of organisms
with natural reproduction resulting in viable and fertile offspring (e.g.
Mayr, 1940). The problem with this concept is that it only applies to
sexually reproducing organisms and it needs proof of successful repro-
duction which is difcult if animals are not collected alive and bred in
the laboratory. Most faunistic studies collecting specimens in the eld
and investigating afterwards the preserved specimens in the laboratory
will not be able to apply the biological species concept and must rely on
other concepts. Applied taxonomy mostly uses the morphological spe-
cies concept which denes a species as a group of individuals that show
the same morphological characteristics and thus can be delimitated from
other groups on the base of morphological differences (e.g. Ax, 1984).
However, there are certain cases in which morphological characters
alone do not allow reliable determination and hence challenge taxono-
mists. The phenomenon of morphological conformity in genetically,
ecologically or otherwise recognizable lineages is known as cryptic di-
versity. Cryptic species are dened as species that are difcult to
distinguish using traditional morphology-based taxonomic methods
(Knowlton, 1993), or species that are classied as a single nominal
species because they are at least supercially morphologically identical
(Bickford et al., 2007). The biological species or other concepts may still
apply to these taxa but not the morphospecies concept, which brings us
back to the discussion about the denition of a species. Among bi-
ologists, there are varying opinions as to the existence of cryptic species.
* Corresponding author.
E-mail address: tobias.p (T. Pngstl).
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Received 2 February 2021; Received in revised form 16 June 2021; Accepted 24 June 2021
Molecular Phylogenetics and Evolution 163 (2021) 107240
Some scientists claim that cryptic species are nothing more than an in-
compatibility of species concepts (e.g. Heethoff, 2018) because the term
‘crypticonly refers to morphology and some concepts do not include the
morphological aspect at all. Indeed, prioritizing one species concept
over the other may result in cryptic diversity, i.e. according to the one
concept there is only one species while according to another concept
there may be more (Heethoff, 2018). There are many other biological
features of an organism that may be responsible for reproductive
isolation, as for example ecological, biochemical, acoustic characters,
and thus may be used for species delimitation. The relative importance
of each biological character for a species concept is again a product of
the human mind and therefore, it is the focus on morphology that creates
‘cryptic speciesas dened above by Knowlton (1993) or Bickford et al.
(2007). Nevertheless, the phenomenon of high phenotypic similarity
despite restricted or even a complete lack of gene ow is real and could
be well demonstrated in various cases (e.g. Fennessy et al., 2016; Struck
et al., 2018b; Sch¨
affer et al., 2019). Even though cryptic species may be
an articial construct, the evolutionary processes leading to phenotypic
similarity are not and should be further investigated. Studying these
cases may allow us to better understand evolutionary processes like
parallelism, convergence and stasis (Struck et al., 2018a, Struck and
Cerca, 2019). To investigate these cases in a proper way, it is necessary
to establish these taxa in our classication system, which means they
should be named and classied because otherwise, biological data will
lose value as it is linked to unnamed vague biological groups and sci-
entists will not be sure if they are talking about the same taxon (Pante
et al., 2015). Moreover, taxa need to be named for being included in
conservation programs (Deli´
c et al., 2017) and no matter if reproduc-
tively isolated species show diverging morphologies or not, they need
the same attention in terms of conservation.
Research on cryptic diversity has intensied over the last two de-
cades and the existence of cryptic species was demonstrated in various
animal groups, as for example in hydrozoans (Holland et al., 2004),
arachnids (Crews and Gillespie, 2010; Knee et al., 2012; McHugh et al.,
2014; Pngstl et al., 2014; Dziki et al., 2015; Sch¨
affer and Koblmüller,
2020), insects (Hebert et al., 2004; Williams et al., 2006; Zangl et al.,
2021), crustaceans (Lee, 2000; Lef´
ebure et al., 2006; Belyaeva and
Taylor, 2009), amphibians (Stuart et al., 2006), reptiles (Smith et al.,
2011) and sh (Colborn et al., 2001; Wagner et al., 2019). Due to the
increasing use of integrative approaches and advanced methods, the
number is continuously growing (Knee et al., 2012). Nevertheless, many
authors refrain from giving cryptic taxa species names, others do, but
fail to explain what species concept was used to erect the new taxon. The
latter further fuels the conict about cryptic species being valid species,
therefore, describing a cryptic species should always be based on a
specic species concept, so that it can be treated like any other species.
In this way, research on the evolutionary processes causing hidden di-
versity is performed in a common system of reference that allows better
Despite the recent increase of detected cryptic species, the underly-
ing evolutionary mechanisms are still poorly understood. In theory,
several processes could lead to the evolution of cryptic species. First,
recent speciation may result in phenotypic similarity as detectable
morphological traits have yet to appear (Holland et al., 2004). Second,
extreme or homogeneous habitat conditions might impose stabilizing
selection on morphology, resulting in highly conserved morphological
traits (Colborn et al., 2001; Lef´
ebure et al., 2006; Bickford et al., 2007).
And third, evolutionary convergence and parallelism may also result in
similar morphotypes across distantly related lineages (Holland et al.,
2004; Belyaeva and Taylor, 2009; Struck and Cerca, 2019).
Regardless of biological denition and evolutionary processes,
cryptic diversity concerns specialists in a broad range of scientic elds
and is supposed to be responsible for gross underestimates of biodiver-
sity in various taxa (Bickford et al., 2007; Pfenninger and Schwenk,
2007; Skoracka et al., 2015). Cryptic species are almost evenly distrib-
uted among major metazoan taxa and biogeographic areas when
corrected for study intensity, and thus the phenomenon of hidden di-
versity is even thought to represent an evolutionary constant (Pfen-
ninger and Schwenk, 2007).
Although cryptic speciation (meaning speciation without morpho-
logical diversication) is supposed to occur in all biogeographic regions
(Pfenninger and Schwenk, 2007), some authors argue that tropical
rainforests and marine habitats represent key targets for investigating
this phenomenon, because they are the most species rich habitats on the
globe and thus the probability of nding cryptic species is also higher
(Holland et al., 2004; Bickford et al., 2007). The Caribbean, which offers
both, lush tropical rainforest and pristine marine habitats, is known to
harbor several cryptic species complexes. Examples are the orb-weaver
spider Micrathena, in which six nominal species consist of eight diver-
gent genetic lineages that are probably all single island endemics
(McHugh et al., 2014), the skipper buttery Astraptes fulgerator, which
has long been regarded as a single species but then was suggested to
contain ten separate species (Hebert et al., 2004), or the cobweb spider
Spintharus avidus, previously presumed to be a single widespread spe-
cies that turned out to represent a complex of at least 16 different species
(Dziki et al., 2015) and the intertidal mite Carinozetes mangrovi, that
shows a trans-Caribbean distribution but consists of three distinct ge-
netic lineages (Pngstl et al., 2019a). This diversity is due to the com-
plex geological history of the Caribbean area, characterized by
continental islands, which broke off from the mainland, land-bridge
islands, which were connected to the continent, uplifted limestone
islands and volcanic islands (Iturralde-Vinent, 2006). In general,
Caribbean biota show high levels of endemism and only a relatively
small percentage of the Caribbean biodiversity is represented by wide-
spread species, presumably taxa with excellent dispersal abilities (Dziki
et al., 2015). Accordingly, allegedly widespread Caribbean taxa that are
poor dispersers are likely to contain hidden species complexes as shown
in the examples mentioned above.
Thalassozetes barbara Pngstl, 2013, a small intertidal sexually
reproducing mite, may in fact represent such a case and thus may serve
as excellent case study to investigate the causes of cryptic diversity and
to demonstrate how to classify such taxa to make them available and
comparable with other non-cryptic taxa. This species is a member of the
intertidal oribatid mite family Selenoribatidae, which has successfully
colonized the extreme intertidal environment of subtropical and tropical
shorelines, where it feeds on marine associated algae (Pngstl, 2017).
These animals are still air breathing but use elaborate plastron respira-
tion to withstand daily tidal ooding (Pngstl and Krisper, 2014).
Thalassozetes barbara was the rst ofcially described intertidal mite
species of the whole Caribbean and was found on the coast of Barbados
(Pngstl, 2013a). Over the last decade subsequent records were made at
various Caribbean locations resulting in a theoretical distribution
pattern ranging from the Bahamas to the Greater Antilles and nally to
the Lesser Antilles close to the coast of South America (Pngstl, 2021).
Given the very small size (approx. 0.3 mm), the wingless body and the
complete lack of active dispersal behavior of these intertidal arthropods,
this wide distribution is puzzling and difcult to explain.
However, littoral oribatids are able to survive submerged in seawater
for even more than a month (Pngstl, 2013b). Accordingly, long dis-
tance transport is suggested to happen mainly by hydrochory (Schatz,
1991; Pngstl, 2013b; 2017), i.e. drifting along ocean currents. In fact,
Thalassozetes balboa Pngstl, Lienhard & Baumann, 2019, a recently
discovered species, was found in Panama as well as in Florida and hence
may show a vast distribution range in the western Caribbean. Addi-
tionally, it was suggested that transport along the Gulf Stream could
have facilitated dispersal along the Central American shoreline (Pngstl
et al., 2019b). The allegedly wide distribution of the Eastern Caribbean
T. barbara could also be the result of hydrochory, but the frequency of
drifting events as well as the real probability of effective gene-ow be-
tween populations of distant islands is unknown and hence the status of
T. barbara as a single nominal species remains questionable.
Here, we present a comprehensive morphometric and molecular
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
genetic investigation of Caribbean T. barbara populations to clarify and
assess the amount of diversity hidden within this group and to provide
new distribution patterns and rst extensive phylo- as well as biogeo-
graphic insights into this group of intertidal Caribbean arthropods.
Additionally, we emphasize the importance of naming cryptic taxa and
propose a possible way of how to describe such species in practice.
2. Material and methods
2.1. Sample collection and preparation
In February 2016 and February 2017, animals were collected on two
different eld trips to selected Caribbean areas. Samples of intertidal
algae (patches of approx. 10 cm
) were scraped off the substrate (e.g.
rock, mud, mangrove roots etc.) with a knife during low tide and put in a
Berlese-Tullgren funnel to extract the mites. Extracted specimens were
stored in >99% ethanol for transport and further investigation. A
complete list of sample locations is given in the Appendix A.
2.2. PCR and sequencing
In total, 117 specimens of Caribbean Thalassozetes spp. were ana-
lysed (see Appendix A). Total genomic DNA was extracted from single
individuals preserved in >99% ethanol. Extraction was carried out
using a Chelex-based method (Casquet et al., 2012) with some adjust-
ments for small arthropods (Lienhard and Sch¨
affer, 2019). Samples were
extracted for 34 hr at 56 C. Three gene fragments were sequenced for
this study: the mitochondrial cytochrome c oxidase subunit 1 gene (COI; N
=117), the nuclear elongation factor 1 alpha gene (EF-1
; N =55), and
the nuclear 18S rRNA gene (18S; N =42). Even though PCR ampli-
cation of the nuclear markers failed in part of the samples, all main
lineages/islands are also represented in the nuclear data. A 567 bp
fragment of the COI gene was amplied using the primer pairs Mite COI
2F and Mite COI 2R (Otto and Wilson, 2001), and for amplifying 513 bp
of the EF-1
gene, the primers 40.71F and 52.RC (Regier and Shultz,
1997) were used. The complete 18S (~1.8 kb) was amplied in two
overlapping fragments according to the PCR protocol of (Dabert et al.,
2010), using the recommended primers (Skoracka and Dabert, 2010).
PCR conditions for the COI gene fragment are given in (Pngstl et al.,
2014) and those for the EF-1
gene fragment in (Lienhard et al., 2014).
DNA purication with ExoSAP-IT (Affymetrix), cycle sequencing using
the BigDye Sequence Terminator v3.1 Cycle Sequencing chemistry
(Applied Biosystems) and cycle sequencing product purication with
Sephadex G 50 (FE Healthcare) were conducted following (Sch¨
et al., 2008). Sequencing was performed in both directions and se-
quences were visualized on an automated capillary sequencer (ABI
PRISM 3130xl, Applied Biosystems). All sequences obtained from this
study were deposited in GenBank (;
accession numbers for COI: MZ169923-MZ170038, EF-1
: MZ220224-
MZ220278, and 18S: MZ220279-MZ220318; more details are given in
the Appendix A).
2.3. Phylogenetic analysis
Electropherograms were checked by eye and sequences were aligned
using MUSCLE (Edgar, 2004) as implemented in MEGA6 (Tamura et al.,
2013), employing the default settings. Gene fragments were analyzed
individually and as a concatenated dataset comprising mtDNA and
nucDNA (COI, EF-1
and 18S, 2882 bp). The best tting models of
molecular evolution were selected based on the Bayesian Information
Criterion (BIC) in Modelnder (Kalyaanamoorthy et al., 2017) (COI,
HKY +I; EF-1
, HKY +I; 18S, HKY +I). For all gene fragments and the
concatenated dataset (partitioned by gene), both a Bayesian inference
(BI) and Maximum Likelihood (ML) tree were inferred in MrBAYES 3.1.2
(Ronquist and Huelsenbeck, 2003) and RAxML (Stamatakis, 2014) via
raxmlGUI 2.0.0 (Endler et al., in press), respectively. MrBayes analyses
applied a MC
simulation with 10 million generations (2 independent
runs, 6 chains, 25% burn-in, best tting substitution model for each
gene). The average standard deviation of split frequencies (<0.01 in all
analyses) was used to assess whether runs were run long enough. In
addition, the resulting log-les were analysed in Tracer 1.6 (Rambaut
and Drummond, 2007) to check for convergence and to ensure statio-
narity of all parameters. RAxML was run using the ML +rapid bootstrap
setting with the GTRGAMMA substitution model (for all genes) 10,000
bootstrap replicates.
2.4. Species delimitation
Single-locus species delimitation was performed by applying
different approaches on the full COI dataset (117 specimens): (1) the
distance based Automatic Barcode Gap Discovery method (ABGD;
Puillandre et al., 2012), and the tree-based (2) Bayesian Poisson Tree
Processes model (bPTP; Zhang et al., 2013), (3) single threshold general
mixed Yule coalescent model (sGMYC; Pons et al., 2006), and (4) the
Bayesian general mixed Yule coalescent model (bGMYC; Reid and
Carstens, 2012). In addition, we employed a Bayesian multilocus species
delimitation method, based on all our three loci, as implemented in
Bayesian Phylogenetics and Phylogeography (BPP 4.1; Rannala and
Yang, 2003; Yang and Rannala, 2010)
The ABGD analysis was conducted via the ABGD web server (http
:// We used sim-
ple distances and ran ABGD under following parameter settings: pmin =
0.005, pmax =0.1, X (relative gap width) =1.0, no. of steps =20. We
recorded the hypothetical species assignments over 20 recursions.
The bPTP analysis was conducted via the bPTP web server (https
://, applying 500,000 MCMC generations and
using the BI tree as input tree.
The sGMYC analysis was conducted on the GMYC web server (htt
ps:// GMYC requires and ultrametric input
tree. Therefore, the BEAST 2.5.1 package (Bouckaert et al., 2014) was
used to infer an ultrametric tree. We ran two MC
simulation with 200
million generations, sampling every 1000th tree (of which 10% were
discarded as burn-in), applying the best-tting substitution model, a
birthdeath tree prior, and a strict clock (a clock-like evolution could not
be rejected at the 0.05 signicance level by means of likelihood ratio
tests in TREE-PUZZLE 5.3; Schmidt et al., 2002). Tracer 1.6 was used to
verify the chains had reached stationarity. Treeles were combined
using LogCombiner (implemented in the BEAST2 package) and
TreeAnnotator (implemented in the BEAST2 package) was used to
calculate a maximum clade credibility (MCC) tree from the post burn-in
tree sample. A single-threshold was employed for GMYC analysis due to
its generally better performance in delimitating species as compared to
the multi-threshold GMYC approach (Fujisawa and Barraclough, 2013).
The bGMYC analysis was conducted on 500 posterior trees from the
BEAST analysis and run (MCMC =50,000; burnin =40,000; thinning =
100) in R v3.6.0 (R Core Team, 2013) using the package bGMYC v.1.0.2
(Reid & Carstens 2012).
For the BPP analysis, the heredity scalar was set to 0.25 and 1.0 for
the mitochondrial and nuclear loci, respectively. Multiple unguided (i.e.
without a guide tree; Rannala and Yang, 2017) analyses were run with
varying prior combinations for ancestral population size (θ) and diver-
gence time (
) (Leache and Fujita, 2010), to account for the effects of
prior settings on the number of inferred species (different congurations
assume different levels of gene tree discordance): i) large ancestral
population size and deep divergence (θ ~ IG (3, 0.4),
~ IG (3, 0.4)); ii)
large ancestral population size and intermediate divergence (θ ~ IG (3,
~ IG (3, 0.04)); iii) large ancestral population size and shallow
divergence (θ ~ IG (3, 0.4),
~ IG (3, 0.004)); iv) intermediate ancestral
population size and deep divergence (θ ~ IG (3, 0.04),
~ IG (3, 0.4)); v)
intermediate ancestral population size and intermediate divergence (θ
~ IG (3, 0.04),
~ IG (3, 0.04)); vi) intermediate ancestral population
size and shallow divergence (θ ~ IG (3, 0.04),
~ IG (3, 0.004)); vii)
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
small ancestral population size and deep divergence (θ ~ IG (3, 0.004),
~ IG (3, 0.4)); viii) small ancestral population size and intermediate
divergence (θ ~ IG (3, 0.004),
~ IG (3, 0.04)); and ix) small ancestral
population size and shallow divergence (θ ~ IG (3, 0.004),
~ IG (3,
0.004)). The prior assignment of individuals to a maximum number of
hypothesized species was based on the identied mitochondrial lineages
/ maximum number of species delimited based on the COI data. The
analyses were run twice for 50,000 generation (following a burn-in of
10,000 steps), sampled every 10 steps.
For all markers, maximum intraspecic and minimum interspecic
distances, based on uncorrected p-distances, were calculated using
SPIDER 1.5.0 (Brown et al., 2012) in R v.3.6.0. SPIDER was also used to
identify diagnostic nucleotides in the COI gene.
2.5. Species tree
A multispecies coalescent analysis based on all three loci was con-
ducted in StarBEAST2, implemented in BEAST2 version 2.5.1 (Bouck-
aert et al., 2014). Eight putative species (see discussion for why we
assigned the samples to these eight putative species) were predened
and data was partitioned by gene (best-tting substitution models COI:
HKY +I; EF-1
: HKY +I; 18S: HKY +I). As likelihood ratio tests in
TREE-PUZZLE 5.3.rc16 (Schmidt et al., 2002) did not reject a clock-like
evolution for any of the three loci, we applied the strict-clock model for
all three genes. The Birth-Death model was selected as tree prior. Three
independent replicates were run with random starting seeds and 2x10
generations, sampled every 20,000 generations, and discarding the rst
10% as burn-in. The effective sample sizes (ESS) of parameters were
checked in Tracer, runs were combined using LogCombiner (part of the
BEAST2 package), and the species tree was visualized as a cloudogram
in DensiTree2 (part of the BEAST2 package).
2.6. Intra-island diversity
To infer the genetic structure within main lineages, statistical
parsimony haplotype networks based on the COI data were inferred
using the program PopART (Leigh and Bryant, 2015), applying the
default settings.
2.7. Morphometric investigations
Specimens were embedded in lactic acid for temporary slides and
measurements were performed using a compound light microscope
(Olympus BH-2) and ocular micrometer. Twenty continuous variables
(Supplementary Fig. S1) were measured in 91 presumed Thalassozetes
barbara individuals from six Caribbean islands (Barbados, Curaçao,
Grenada, Martinique, Guadeloupe and New Providence Island/
Bahamas) and in 46 T. balboa specimens from Central America (Pan-
ama). As specimens were destroyed for DNA-extraction, these speci-
mens, used here for morphometry, were different individuals but
originated from the exact same samples (10 cm
patch of algae),
meaning they belonged to the same population. There were not enough
specimens from the Dominican Republic available for morphometric
investigation, therefore this material was only used for molecular ge-
netic studies.
Non-Metric Multidimensional Scaling (NMDS, based on Euclidian
distances, two-dimensional) and Linear Discriminant Analysis (LDA)
was performed on log
-transformed raw and size-corrected data to
reveal possible differences between the populations and to determine
the most important differentiating variables. Size correction was done
by dividing each variable through the geometric mean of the respective
specimen. For testing the equality of means of all populations, Multi-
variate Analysis of Variance (MANOVA) was used and for pairwise
comparisons, Hotellings T
-tests were conducted.
For the variables contributing most to differentiation between the
supposedly cryptic Thalassozetes (excluding T. balboa which is
morphologically signicantly different) as detected by LDA, univariate
statistics were performed. Mean, minimum, maximum, standard devi-
ation and coefcient of variation (cv) were calculated, and Kruskal-
Wallis and Mann-Whitney U test were used for comparing the means
of variables between all populations and for pairwise comparisons,
respectively. All analyses were performed with PAST 3.11 (Hammer
et al., 2001).
2.8. Morphological investigations
For microscopic investigation in transmitted light, preserved animals
were embedded in Berlese mountant. Depictions were made with an
Olympus BH-2 Microscope equipped with a drawing attachment. These
drawings were rst scanned, then processed and digitized with the free
and open-source vector graphics editor Inkscape (freeware available
For photographic documentation, specimens were air-dried and
photographed with a Keyence VHX-5000 digital microscope in reected
Due to the low number of specimens (n =3, 2 adults, 1 juvenile) we
were not able to provide any depiction of the cryptic Thalassozetes
species from the Saman´
a Peninsula (Dominican Republic). The two
adults were used for molecular genetic analyses and hence partly
destroyed but microscopic investigations of the remains conrmed the
identical morphology with the other members of the cryptic species
3. Results
3.1. Phylogenetic analyses
For all datasets, Bayesian Inference (BI) and Maximum Likelihood
(ML) analyses produced congruent phylogenies. Resolution differed
among the datasets. Most nodes connecting the main lineages were well
resolved in the trees based on COI and the concatenated dataset (Figs. 1
& 2). Ten main lineages, largely corresponding to distinct geographic
regions, were recovered in these trees: i) Panama and Florida, ii)
Bahamas, iii) Curaçao and southern coast of Dominican Republic, iv)
northern coast of Dominican Republic, v) Martinique, vi) Guadeloupe,
vii) Barbados, viii x) three lineages from Grenada, including two
samples from Barbados. In contrast, the two nuclear markers produced
only poorly resolved trees, with only some of the mitochondrial lineages
recovered as distinct lineages also in these trees (Supplementary
Fig. S2). In both nuclear datasets, however, both samples from Panama
and the Bahamas were resolved as quite divergent from the rest. In
addition, Curaçao plus southern Dominican Republic, Barbados and
northern Dominican Republic plus Guadeloupe, resulted as somewhat
distinct in the EF-1
tree (Supplementary Fig. S2A).
3.2. Species delimitation
Results from single locus species delimitation methods were incon-
gruent, especially between distance- and tree-based approaches, but all
methods identied 7 putative species (Fig. 1). ABGD recognized seven
distinct Thalassozetes species at an initial partition with intraspecic
divergence <5%: i) Panama and Florida (T. balboa), and with the
T. barbara complex ii) Bahamas, iii) Curaçao and southern coast of
Dominican Republic, iv) northern coast of Dominican Republic, v)
Martinique and Guadeloupe, vi) Barbados, viii) Grenada. At an initial
partition with intraspecic divergence <2.5%, ABGD identied 11
species, by splitting samples from Martinique and Guadeloupe into two
distinct species, and further assigning samples from Grenada to three
distinct entities. GMYC detected 15 species with high support values
(>80). Also, the bPTP analysis resulted in a best supported partition of
15 (1121) species, as well as bGMYC, when assuming a rather con-
servative probability threshold (posterior probability: 0.5 <P <0.9) for
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
Fig. 1. BI tree of Thalassozetes spp. based on the COI data. As measures of nodal support, posterior probabilities (from BI; only values >0.7) and bootstrap support
values (from ML tree inference; only values >50) are shown. For samples highlighted in grey, only COI data are available. Bars and heatmap to the right indicate the
number of putative species inferred by different single-locus species delimitation methods.
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
identifying putative species, compared to higher thresholds that could
overestimate the species number (Kornilios et al. 2020). These three
tree-based methods identied additional species in Panama, the
Bahamas, Martinique, Guadeloupe and Barbados. Some of these putative
species comprise only a single sample, characterized by a rather long
branch length. Indeed, long branch lengths might be indicative for
alignment errors or pseudogenes, but a careful double check (including
translation into an amino acid sequence) suggested these sequences
were okay and not pseudogenes.
Multilocus species delimitation using BPP favored, depending on the
prior settings, a different number of putative species (Fig. 2). All prior
combinations with small and intermediate ancestral population sizes
strongly favored the existence of 12 putative species, corresponding to
the main clades in the phylogenetic tree and two singletons (from
Martinique and Guadeloupe) with slightly longer branch lengths. Prior
combinations that included large ancestral population sizes, on the other
hand, yielded roughly equal support for 9 or 10 putative species.
However, the 10 species scenario mainly consisted of two equally well
supported models (posterior probability of ~ 0.2 for each of these two
models), while a single model contributed most to the 9 species scenario.
The inferred putative species again largely corresponded to the main
clades in the phylogenetic tree, but the 9 species model grouped
northern Dominican Republic and Guadeloupe together as one species,
which was also the case for the second 10 species model that also
identied a singleton from Martinique as distinct species.
In general, the various molecular species delimitation methods sug-
gested a minimum of seven and a maximum of 15 distinct species. Based
on these results and on morphometric and distribution data (see below),
we postulate eight Caribbean Thalassozetes species including six yet
undescribed taxa. The latter will be referred to in the following text with
their names: Thalassozetes grenadensis sp. n., Thalassozetes dushi sp. n.,
Thalassozetes guadeloupensis sp. n., Thalassozetes martiniquensis sp. n.,
Thalassozetes paradisi sp. n. and Thalassozetes samanae sp. n.
Minimum interspecic COI distances among the proposed eight
species range from 5.4 to 16.3% (maximum of 12.4% among the island
taxa), and always exceeded maximum intraspecic distances in these
species, indicating a clear barcoding gap (Fig. 3a,d). In the nuclear data,
no barcoding gap was observed (Fig. 3b,c), due to the much lower
Fig. 2. Multi-locus species delimitation in the genus Thalassozetes. (a) BI tree showing the phylogenetic relationships among Thalassozetes spp. based on the
concatenated datasets. As measures of nodal support posterior probabilities (from BI; only values >0.7) and bootstrap support values (from ML tree inference; only
values >50) are shown. Bars to the right indicate the best supported species hypotheses inferred by BPP under various prior settings as shown in (b) and the nal
conservative species delimitation considering not only molecular, but also morphological and geographic evidence.
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
substitution rates of these markers as compared to the mitochondrial
COI gene.
3.3. Species tree
The topology of the species tree (Fig. 4) based on all three loci (COI,
, 18S) is different from the phylogenetic tree inferred from the
concatenated dataset (Fig. 2), which is to be expected as the tree to-
pology from the concatenated dataset is heavily driven by the COI data,
which shows much more variation than the two nuclear markers. Again,
the mainland species T. balboa resulted as sister species of the Caribbean
taxa. Within this species complex, T. paradisi sp. n. (Bahamas) was
resolved as sister taxon of the remaining Caribbean species. Phyloge-
netic relationships among these were largely poorly supported, with the
exception of a monophylum comprising T. martiniquensis sp. n.
(Martinique) and T. guadeloupensis sp. n. (Guadeloupe) and
T. grenadensis sp. n. (Grenada), which were resolved as sister species
with high statistical support.
3.4. Phylogeographic structure and distribution patterns on islands
The haplotype network for the mainland species T. balboa shows no
clear phylogeographic structure with a generally high haplotype di-
versity and haplotype sharing between the sampling sites in Florida and
Panama (Fig. 5a). In contrast, the Caribbean island species do show,
sometimes pronounced, phylogeographic structure, even on compara-
tively small islands (Fig. 5b, 6). Low levels of haplotype sharing suggest
occasional gene ow between geographically distant localities on these
Fig. 3. Comparison of maximum intraspecic and minimum interspecic distances among Thalassozetes spp. (a) COI. (b) EF-1
. (c) 18S. (d) Barcode gap plot
showing the minimum interspecic vs. the maximum intraspecic p-distance based on the COI data. Dots above the 1:1 line indicate the presence of a barcode gap.
None of the species exhibits higher intraspecic than interspecic divergence.
Fig. 4. Multispecies coalescent tree of the genus Thalassozetes. The consensus
phylogeny is superimposed on a DensiTree cloudogram of alternative sampled
trees, with contrasting topologies highlighted by different colors. Nodal support
in form of posterior probabilities of 0.90 0.99 is indicated by grey and
black circles, respectively (only values >0.7 are shown).
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
Fig. 5. Statistical parsimony haplotype net-
works based on COI sequences. Each circle
corresponds to one haplotype and its size is
proportional to its frequency. The number of
mutations is indicated as hatch marks. Small
black circles represent intermediate haplo-
types not present in the dataset. Colors refer to
different locations/islands as indicated on the
respective map. (a) Thalassozetes balboa from
Panama and Florida. (b) Thalassozetes paradisi
sp. n. haplotypes from New Providence Island,
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
islands (e.g. Barbados, Fig. 6a, Grenada, Fig. 6b). The two quite diver-
gent haplotypes on Barbados belong to a different species, T. grenadensis
sp. n., otherwise only found on Grenada.
3.5. Morphometry
Excluding the present cryptic taxa, the genus Thalassozetes comprises
ve morphospecies and is mainly characterized by the presence of
lamellar ridges, a clavate sensillum and 1314 notogastral setae
(Pngstl 2013a). These ve species can be distinguished by the pattern
of notogastral cuticular structure, number of notogastral ridges, shape of
epimeral cavity and the presence of proximoventral teeth on claws. The
present Caribbean island taxa, on the other hand, show complete con-
formity in all these diagnostic characters with T. barbara and lack
additional distinctive features allowing to distinguish between them.
Although these taxa cannot be distinguished based on distinct
morphological characters, they do differ in morphometric characteris-
tics. In accordance with the results of the molecular genetic analyses, the
populations of each island will subsequently be labelled with their
respective new species names.
LDA conducted on both raw and size-corrected data revealed that
mites from each island, show diverging clusters, whereas some of the
cluster still exhibit overlaps (Fig. 7). The populations from Curaçao
(T. dushi sp. n.), Guadeloupe (T. guadeloupensis sp. n.) and Martinique
(T. martiniquensis sp. n.) are largely overlapping, indicating few
morphometric differences between them. The population from Barbados
(T. barbara) shows fewer overlaps, and the populations from Bahamas
(T. paradisi sp. n.), Grenada (T. grenadensis sp. n.) and Panama
(T. balboa) are clearly separated from all other populations. MANOVA
showed highly signicant differences (p <0.001) between all pop-
ulations in raw as well as size-corrected data, and pairwise Hotelling‘s
-tests between the populations always revealed signicant differences
(p <0.05). All-samples LDA correctly classied 96.35% (Jackknifed
84.67%) of all specimens in raw and 94.89% (Jackknifed 69.34%) in
size-corrected data. The most important variables responsible for sepa-
ration, gained by LDA, were db, ll, dnr, efw1, efw2 and gl, which means
the posterior prodorsal, the anterior notogastral area and the epimeral
foveae are mainly affected (Supplementary Table S1).
Fig. 6. Statistical parsimony haplotype networks based on COI sequences. Each circle corresponds to one haplotype and its size is proportional to its frequency. The
number of mutations is indicated as hatch marks. Small black circles represent intermediate haplotypes not present in the dataset. Colors refer to different locations/
islands as indicated on the respective map. (a) Thalassozetes barbara populations from Barbados. (b) Thalassozetes grenadensis sp. n. populations from Grenada.
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
These variables, as well as body length (bl) and width (dened by
), were subsequently analyzed by univariate statistics and showed
signicant differences between all populations, cryptic species respec-
tively, as well as in several pairwise comparisons (Table 1). However, no
reliable variables for species delimitation could be dened as almost all
variables overlap when their whole range is considered. The only
exception can be found in the population from Bahamas (T. paradisi sp.
n.), as its values for db (distance between bothridia) are always smaller
than in the other populations and its values for ll (lenticulus length) are
always higher. Still, these differences are minute and can thus not be
considered as reliable for species delimitation, either.
3.6. Taxonomy and morphology
As all species show conformity in their morphology with Thalasso-
zetes barbara, the original detailed description and the diagnosis given
by Pngstl (2013a) are valid for all members of this cryptic species
complex. A slightly updated version of this diagnosis is available in the
supplementary material (Supplementary Text le S1). Herein, we only
provide depictions of each species and information about slightly
diverging non-diagnostic characters, if present (e.g. body size, surface
Family Selenoribatidae Schuster, 1963
Genus Thalassozetes Schuster, 1963
Type species: Thalassozetes barbara Pngstl, 2013
Thalassozetes dushi sp. n.
Types: Holotype - Curaçao, Boca Ascenci´
on, from Bostrychia growing
on intertidal rock, 5 Feb. 2016; preserved in ethanol, deposited at the
Naturhistorisches Museum Wien (Vienna). Paratypes - four specimens,
same location as holotype, preserved in ethanol, deposited at the US
National Museum collection (USDA-Beltsville, MD).
Type locality: Curaçao, Boca Ascenci´
on; Lesser Antilles
GenBank accession numbers: COI: MZ169923MZ169929, EF-1
MZ220224MZ220229, 18S: MZ220313MZ220318
Molecular diagnosis: In our COI alignment, position 3 is occupied by
base C, position 27 by base C, position 123 by base G, position 135 by
base G, position 360 by base C, position 411 by base G, position 516 by
base C, and position 549 by base A (Supplementary Table S2).
ZooBank registration:
Etymology: The specic epithet is the Papiamentu (Creole language)
word dushi, which means charming or cute. As this eight-legged species
may not look cute or charming to most of us, the word dushi rather refers
to Curaçao, the type locality, where people often use this adjective to
describe the island. Here it is given as noun in apposition.
Distribution: Curaçao, Hispaniola (Dominican Republic) (see Fig. 8).
Morphological remarks: Body length 277297 µm, body width
135160 µm (n =10) (Fig. 9, Supplementary Fig. S3).
Thalassozetes grenadensis sp. n.
Types: Holotype - Grenada, La Sagesse Bay, from Bostrychia growing
on intertidal rock, 27 Feb. 2016; preserved in ethanol, deposited at the
Naturhistorisches Museum Wien (Vienna). Paratypes - four specimens,
Grenada, Devils Bay, from green intertidal algae on limestone rock, 28
Feb. 2016; preserved in ethanol, deposited at the US National Museum
collection (USDA-Beltsville, MD).
Type locality: Grenada, La Sagesse Bay; Lesser Antilles
GenBank accession numbers: COI: MZ169932MZ169967, EF-1
MZ220232MZ220260, 18S: MZ220395MZ220311
Molecular diagnosis: In our COI alignment, position 15 is occupied
by base A or G, position 51 by base C, position 426 by base T, and po-
sition 528 by base C (Supplementary Table S2).
ZooBank registration:
Etymology: This species is named after the Lesser Antillean island
Grenada, the type locality of this species.
Distribution: Grenada, Barbados western coast (see Fig. 8).
Morphological remarks: Body length 280317 µm, body width
166191 µm (n =36). Prodorsal ridges not as prominent as in T. barbara
(Fig. 9, Supplementary Fig. S3). Notogastral ridges variable in height
and shape.
Fig. 7. Scatter plots gained from Non-Metric Multidimensional Scaling (left side) and Linear Discriminant Analysis (right side) on raw data (upper row) and size-
corrected data (lower row). Overlapping clusters indicate morphological similarity and displaced clusters reect diverging body shapes.
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Molecular Phylogenetics and Evolution 163 (2021) 107240
Thalassozetes guadeloupensis sp. n.
Types: Holotype - Guadeloupe, Capesterre-Belle-Eau (Basse-Terre),
from Bostrychia on intertidal rock, 19 Feb. 2016; preserved in ethanol,
deposited at the Naturhistorisches Museum Wien (Vienna). Paratypes -
four specimens, same location as holotype, preserved in ethanol,
deposited at the US National Museum collection (USDA-Beltsville, MD).
Type locality: Guadeloupe, Capesterre-Belle-Eau (Basse-Terre);
Lesser Antilles
GenBank accession numbers: COI: MZ169968MZ169975, EF-1
MZ220261MZ220266, 18S: MZ220289MZ220294
Molecular diagnosis: In our COI alignment, position 213 is occupied
by base G, and position 408 by base A (Supplementary Table S2).
ZooBank registration:
Etymology: The species name refers to the type locality, the Antillean
island of Guadeloupe.
Distribution: Guadeloupe - endemic (see Fig. 8).
Morphological remarks: Body length 280317 µm, body width
166175 µm (n =12). Notogastral ridges less prominent than in
T. barbara but stronger than in T. paradisi sp. n. (Fig. 9, Supplementary
Fig. S3).
Thalassozetes martiniquensis sp. n.
Types: Holotype - Martinique, Trinit´
e, from Bostrychia growing on
intertidal rock, 24 Feb. 2016; preserved in ethanol, deposited at the
Naturhistorisches Museum Wien (Vienna). Paratypes - four specimens,
Martinique, Pointe du Bout, from Bostrychia on conglomerate rock, 22
Feb. 2016; preserved in ethanol, deposited at the US National Museum
collection (USDA-Beltsville, MD).
Type locality: Martinique, Trinit´
e; Lesser Antilles
GenBank accession numbers: COI: MZ169976MZ169980, EF-1
MZ220267MZ220270, 18S: MZ220285MZ220288
Molecular diagnosis: In our COI alignment, position 96 is occupied
by base T (Supplementary Table S2).
ZooBank registration:
Etymology: The specic epithet refers to the type locality, the
Antillean island of Martinique.
Distribution: Martinique endemic (see Fig. 8).
Morphological remarks: Body length 277308 µm, body width
160182 µm (n =8) (Fig. 9, Supplementary Fig. S3).
Thalassozetes paradisi sp. n.
Types: Holotype - Bahamas, Paradise Island, from Bostrychia growing
on littoral rock, 18 Feb. 2017; preserved in ethanol, deposited at the
Naturhistorisches Museum Wien (Vienna). Paratypes - four specimens,
Bahamas, New Providence Island, Compass Point, from Bostrychia
growing in rock crevice, 19 Feb. 2017; preserved in ethanol, deposited at
the US National Museum collection (USDA-Beltsville, MD).
Type locality: Bahamas, Paradise Island, New Providence.
GenBank accession numbers: COI: MZ170003MZ170013, EF-1
MZ220275MZ220276, 18S: MZ220280MZ220281
Molecular diagnosis: In our COI alignment, position 279 is occupied
by base C, position 342 by base T, position 363 by base G, position 543
by base C, and position 552 by base C (Supplementary Table S2).
ZooBank registration:
Etymology: This species is named after Paradise Island, a small island
and part of New Providence Bahamas, where it was originally discov-
ered; the Latin name for paradise is given in the genitive case.
Distribution: Bahamas endemic (see Fig. 8).
Morphological remarks: Body length 280295 µm, body width
160172 µm (n =4). Anterior notogastral ridges weakly developed and
less protruding than in T. barbara and all other cryptic species. Cer-
otegumental layer showing basically stronger and ner granulation than
in all other species (Fig. 9, Supplementary Fig. S3).
Thalassozetes samanae sp. n.
Types: Holotype Dominican Republic, El Portillo, from Bostrychia
Table 1
Univariate statistics for the cryptic Caribbean Thalassozetes species and comparison of the eight most important morphological variables. Min-max =minimummaximum values in µm; sd =standard deviation and cv =
coefcient of variation (marked light grey if equal or higher than 0.10). Results of Kruskal-Wallis Test (KW) and MannWhitney-U test are given; * =p <0.05, ** =p <0.01, ***=p <0.001. a - Grenada vs. Barbados, b -
Grenada vs. Bahamas, c - Grenada vs. Martinique, d - Grenada vs. Curacao, e - Grenada vs. Guadeloupe, f - Barbados vs. Curacao, g - Bahamas vs. Barbados, h - Bahamas vs. Curacao, i - Barbados vs. Guadeloupe, j - Bahamas
vs. Guadeloupe.
Barbados (T. barbara) n =19 Bahamas (T. paradisi) n =4 Curacao (T. dushi) n =10 Guadeloupe (T. guadeloupensis) n =12 Grenada (T. grenadensis) n =36 Martinique (T. martiniquensis) n =8 KW Mann-Whitney-U
Min-max Mean sd cv Min-max Mean sd cv Min-max Mean sd cv Min-max Mean sd cv Min-max Mean sd cv Min-max Mean sd cv
bl 271302 284.7 8.63 0.03 280295 285.3 6.65 0.02 277297 284.4 6.87 0.02 280317 297.6 11.16 0.04 280317 302.3 8.04 0.03 277308 283.5 10.16 0.04 *** a.d ***; d **; i *
163182 170.3 5.62 0.03 160172 164.5 5.20 0.03 157175 165.0 4.94 0.03 166175 171.5 3.09 0.02 166191 179.8 6.69 0.04 160182 165.5 6.87 0.04 *** a. d ***; c.e **; b *
ll 2834 30.5 2.29 0.08 4046 42.3 2.87 0.07 3437 34.6 1.26 0.04 3140 33.3 2.60 0.08 3140 34.7 2.04 0.06 3134 33.1 1.25 0.04 *** a ***; f **; b.g.h *
dnr 3143 36.6 2.65 0.07 3134 31.8 1.50 0.05 3743 41.2 2.10 0.05 3443 38.2 3.10 0.08 3549 40.6 3.48 0.09 3743 40.0 2.27 0.06 *** a ***; f **;b *
2231 25.5 2.29 0.09 2526 25.3 0.50 0.02 2231 27.3 2.50 0.09 2531 27.3 2.19 0.08 2228 25.0 1.76 0.07 2526 25.1 0.35 0.01 ** e *
1925 21.1 2.28 0.11 1519 16.0 2.00 0.13 1922 20.5 1.58 0.08 1922 20.3 1.54 0.08 1525 20.2 2.13 0.11 1622 19.4 2.50 0.13 *
db 4953 51.3 1.33 0.03 4648 47.0 1.15 0.02 4955 51.8 2.10 0.04 4955 52.5 1.93 0.04 5162 55.5 2.90 0.05 4955 51.3 2.12 0.04 *** a ***; b.c.d.e.g.j *
gl 3445 38.3 3.77 0.10 3743 40.0 3.46 0.09 3440 36.1 2.85 0.08 3443 37.7 3.58 0.09 3446 40.9 3.79 0.09 3445 35.5 3.85 0.11 *** c.d *
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Molecular Phylogenetics and Evolution 163 (2021) 107240
Fig. 8. Map showing the geographic distribution of all Caribbean Thalassozetes species. Different colors and numbers refer to different species. Circles represent
members of the cryptic Thalassozetes species complex, squares indicate non-cryptic species.
Fig. 9. Photographic comparison (stacked stereomicroscopic images) of cryptic Thalassozetes species in dorsal view. Scale bar is valid for all photographs. Photo-
graphs show some of the specimens used in the morphometric analysis.
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
growing on mangrove root (Rhizophora mangle), 11 Feb. 2016; preserved
in ethanol, deposited at the Naturhistorisches Museum Wien (Vienna).
Paratypes - two specimens, same location as holotype, preserved in
ethanol, deposited at the Museo Nacional de Historia Natural "Prof.
Eugenio de Jesús Marcano", Dominican Republic.
Type locality: Dominican Republic, El Portillo, Saman´
a; Hispaniola,
Greater Antilles
GenBank accession numbers: COI: MZ169930MZ169931, EF-1
MZ220230MZ220231, 18S: MZ220312
Molecular diagnosis: In our COI alignment, position 288 is occupied
by base G, position 432 by base C, position 453 by base G, position 486
by base T, and position 513 by base C (Supplementary Table S2).
ZooBank registration:
Etymology: This species was only found on coasts of Saman´
a, a
peninsula and province of the Dominican Republic, therefore the spe-
cic epithet refers to this location and is given as noun in the genitive
Distribution: endemic to Hispaniola (Dominican Republic) (see
Fig. 8).
Morphological remarks: Body length 278289 µm, body width
163169 µm (n =2).
Remarks: The species of this cryptic complex can be distinguished
from the Caribbean T. balboa by the presence of three adanal setae
(instead of two), by having only one ventral tooth on each leg claw
(instead of two) and by the cuticular notogastral pattern with unevenly
distributed circular depressions resulting in an irregular reticulate-
foveate pattern (vs. evenly distributed depressions resulting in regular
reticulate-foveate pattern).
4. Discussion
4.1. Cryptic diversity and its causes
Widespread species are improbable taxonomic hypotheses for line-
ages with poor dispersal abilities, as for example ightless arthropods
(Dziki et al., 2015), and this is particularly true for the Caribbean mite
Thalassozetes barbara. Our results demonstrate that this supposedly
widespread species actually represents a complex that includes at least
six additional cryptic groups, nearly all of which are endemic to single
islands. Minimum interspecic COI distances among the proposed eight
species range from 5.4 to 16.3% (maximum of 12.4% within the island
taxa), and were always larger than the maximum intraspecic distances.
In arthropods, a ten percent divergence in COI exceeds known species
delimitation thresholds (Cosgrove et al., 2016) and the values of most
Thalassozetes groups are in accordance with this suggested benchmark.
Moreover, recent studies on cryptic diversity in tree-living oribatid mites
found uncorrected p-distances ranging from 16 to 24.8% (Sch¨
affer et al.,
2019) and 12.719.6% (Sch¨
affer and Koblmüller, 2020) in the COI gene
between eight and six putative species, respectively. Only some of these
species were classied as clear (but morphologically very similar)
morphospecies. However, congruent clustering of individual specimens
in mitochondrial and nuclear single gene trees and syntopic occurrence
of two or more genetic clusters at several locations indicated repro-
ductive isolation and the existence of previously unknown true biolog-
ical species even when morphological differentiation was lacking (at
least in the morphological characters looked at; Sch¨
affer et al., 2019;
affer and Koblmüller, 2020). Reproductive isolation among the
Thalassozetes groups cannot be veried directly, because they do not
occur syntopically and cross-breeding experiments are more or less
unfeasible as these would last for years due to low reproductive rates
and difculties in simulating the intertidal environment in the lab.
Interspecic sequence divergences in the COI lie (with the exception of
T. guadeloupensis sp. n. and T. martiniquensis sp. n.) within the range
(though at the lower edge) previously inferred for other oribatid species
(e.g. Pngstl et al., 2019b; Sch¨
affer et al., 2019; Seniczak et al., 2019;
affer and Koblmüller, 2020). The lack of resolution we see in our two
nuclear markers, that does not permit us to separate all Thalassozetes
species with condence with these markers, is due the more recent
divergence of Thalassozetes as compared to other previously studied
oribatid mite taxa, as indicated by the observed levels of COI divergence.
In morphologically cryptic taxa, molecular approaches have been
widely used to delineate species, but, where a range of methods are
employed on the same dataset, species delimitation results are often
incongruent. Several factors have been shown to affect molecular spe-
cies delimitation analyses, e.g. population size and divergence time (and
the ratio thereof), gene ow, number of species involved, speciation
rate, sample size and geographic coverage per species, or number of loci
(e.g. Dellicour and Flot, 2015; 2018;; Ahrens et al., 2016; Eberle et al.,
2018; Luo et al., 2018). Among the single-locus species delimitation
methods, distance-based methods like ABGD tend to underestimate
species numbers, while tree-based approaches like GMYC, bGMYC and
PTP often oversplit species (e.g. Dellicour and Flot, 2018; Luo et al.,
2018). We observed the same tendencies in our data, with ABGD nding
fewer putative species than GMYC, PTP and bGMYC, mainly because the
latter approaches often identied somewhat divergent singletons as
distinct species. The multilocus-method BPP shows lower rates of spe-
cies overestimation and underestimation, and should be generally more
robust to various potential confounding factors (Luo et al., 2018). BPP,
as a method that employs the multispecies coalescent, however, di-
agnoses genetic structure and not necessarily species, and importantly, it
does not statistically distinguish between structure associated with
population isolation and species boundaries (Sukumaran and Knowles,
2017). Therefore, it is important to interpret the results of molecular
species delimitation, be it based on single-locus approaches or the
multispecies coalescent, together with other lines of evidence, e.g., from
morphology, ecology, geography, or population genetics (Solis-Lemus
et al., 2015). As our nuclear data contain only very limited variation, the
BPP analysis was probably heavily inuenced by the highly variable
mitochondrial data. All scenarios with low to intermediate ancestral
population size priors identied a larger number of species (some sin-
gletons were identied as distinct species) than scenarios assuming large
ancestral population sizes. Notably, most methods, except for ABGD at
the <5% threshold, identied three species on Grenada. The haplotype
network, showed some clear phylogeographic structure with the three
divergent lineages predominant in distinct parts of the island. But, since
we also found low levels of haplotype sharing among these regions, no
difference in the nuclear markers, and no obvious morphometric clusters
that might correspond to the mitochondrial clades, we refrain from
considering these as different species.
Despite several overlaps, morphometric results and clusters coincide
very well with COI data. Considering that the morphologically distinct
T. balboa from Panama is not conspicuously clearer contrasted in mor-
phospace than some of the cryptic taxa, morphometric data provides
additional evidence for the distinctness of each Thalassozetes lineage.
However, morphometric data does neither provide distinct separation of
all cryptic taxa nor does it clearly conform with any of the species de-
limitation analyses, therefore establishing exactly six new species might
appear to be based on weak reasoning. By adding the geographic
component, however, the six new species are well justied. All these
species are poor dispersers, as clearly indicated by genetic data, and thus
can be considered as island endemics. Even though restricted recent
(potentially human-induced) migration between islands was found in
two of the species, it is highly unlikely that strong hybridization or
intermingling events between populations of different islands occur.
Nevertheless, formally designating cryptic species necessitates using
a species concept, other than the morphospecies concept, to determine
species boundaries. However, this may lead to problems with incom-
patible concepts resulting in grouping artifacts (Heethoff, 2018) as
mentioned in the introduction. De Queiroz (2007) argued that, despite
their various differences, there is a common element in all species
concepts and therefore he proposed a unied concept that dened
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
species as separately evolving metapopulation lineages. He suggested
that the presence of any property used in different species concepts, e.g.
reproductive isolation used in the biological concept or decits of ge-
netic intermediates used in the genetic concept, constitutes evidence for
lineage separation and hence for a separate species. We interpret our
results in the sense of this concept and dene the species herein. Despite
the lack of distinct separating morphological traits and evidence of
reproductive isolation, molecular genetic data as well as the geographic
setting with large oceanic barriers between the islands renders the
Thalassozetes taxa as separately evolving metapopulation lineages and
thus conrms the species in the sense of De Queiroz (2007).
From a practical point of view, this means that studies including any
of the Caribbean Thalassozetes species should ideally use molecular ge-
netic data to identify the respective cryptic taxon. However, faunistic
investigations may not always be able to apply these methods due to
high costs or lacking infrastructure. For these cases we recommend to
determine the species based on their geographic origin, i.e. if the species
was found on Grenada it should be identied as T. grenadensis or if it was
found on Guadeloupe it should be identied as T. guadeloupensis. If a
Thalassozetes specimen is found on any other Caribbean location than
investigated in the present study and shows the phenotype of T. barbara,
it should be classied as Thalassozetes cf. barbara until molecular genetic
data allows a more precise identication. The latter may also be applied
when specimens are found on coasts of Hispaniola because at least two
species are present on this Greater Antillean island which prevents using
geographic origin as classication tool.
4.2. Phylogeography and dispersal
Considering the geographic distribution of these cryptic Caribbean
Thalassozetes, with nearly all species being single island endemics,
geographic isolation and associated genetic drift are most likely the
primary cause for speciation. However, the question arises, what caused
the identical phenotypic appearance of this cryptic Thalassozetes species
complex. There are three evolutionary processes that could lead to
phenotypic similarity in historically isolated lineages, namely recent
speciation, evolutionary convergence and stabilizing selection (Colborn
et al., 2001; Lef´
ebure et al., 2006; Bickford et al., 2007; Fiˇ
ser et al.,
2017). Considering the large sequence divergence among the distinct
Thalassozetes species, recent speciation can be excluded. Moreover, DNA
sequence data renders all Caribbean Thalassozetes species a mono-
phyletic group, which clearly contradicts convergence being responsible
for supercially identical morphologies. The observed morphological
stasis is therefore most likely a product of stabilizing selection, imposed
by extreme and homogeneous environments, reducing or eliminating
morphological change that usually accompanies speciation (Bickford
et al., 2007; Lef´
ebure et al., 2006). All of the cryptic Thalassozetes species
dwell in the intertidal zone, which is an extreme environment because
parameters change constantly and animals have to cope with terrestrial
and marine conditions at the same time. Although the species occur on
distant islands, selective constraints are the same in each littoral zone
and therefore each species is subject to the same selective regime pre-
serving the bauplan.
However, different microhabitats may be present in the intertidal
zone and cryptic species may use different ecological niches, as for
example shown in two Bermudian cryptic intertidal mite species of the
genus Carinozetes, where one species dwells predominantly on rocky
shores while the other occurs exclusively in mangrove forests (Pngstl
et al., 2014). This is not the case in Thalassozetes, as all populations were
collected from algae growing on littoral rocks, with only one single
specimen of T. samanae sp. n. found on algae growing on mangrove
roots. The vast majority of populations were extracted from the red alga
Bostrychia tenella, which is used as substrate and food source by the
mites. Though a recent study also demonstrated this alga to represent a
complex of at least three cryptic and closely resembling species (Zuc-
carello et al, 2015), a correlation with cryptic Thalassozetes species may
be excluded because distribution patterns are not in agreement at all.
Mitochondrial COI sequence data, and to a lesser extent also the
nuclear data, suggest that after the radiation of the Caribbean Tha-
lassozetes group nearly all species have evolved in isolation without any
considerable gene ow between the islands. Moreover, the haplotype
networks show strong diversication and phylogeographic structure on
single islands, which indicates restricted gene ow even on a local scale.
Accordingly, these Thalassozetes species are poor dispersers that prob-
ably rely on rare and stochastic hydrochorous transport, i.e. drifting
along ocean currents (e.g. Pngstl, 2017). Other small arthropods, as for
example the cobweb spider Spintharus or the orb-weaver Micrathena also
show high levels of single island or short range endemics in the Carib-
bean (McHugh et al., 2014; Dziki et al., 2015) indicating that limited
overwater dispersal and vicariance is one of the main factors shaping the
evolutionary history of these small organisms. Nevertheless, haplotype
data show that at least two recent dispersal events have happened: rst,
T. dushi sp. n. has successfully crossed the Caribbean Sea between
Curaçao and the Dominican Republic and second, a few T. grenadensis
sp. n. specimens have reached the coasts of Barbados. The former is quite
unusual as the Caribbean Sea stretches over 600 km between these two
locations and thus should represent a large barrier. How gene ow has
nevertheless happened is presently only a matter of conjecture but large
eddies, bird mediated transport or even recent anthropogenic dispersal
could be responsible.
The non-cryptic Western Caribbean Thalassozetes species, T. balboa,
shows a completely different pattern with ongoing gene ow and a
possible wide distribution from Panama to Florida. The occurrence of
this species seems to range across the whole Caribbean Central American
coastline (Pngstl et al., 2019b). Therefore, in this species, dispersal and
exchange between populations may occur along the shore without any
oceanic barriers. Moreover, the Gulf Stream may facilitate dispersal
along the coastline at least in one direction and this together may result
in the obviously diverging biogeographic pattern.
As the open ocean clearly represents a barrier for the species of the
cryptic Thalassozetes complex, the common ancestor of this group sup-
posedly occupied former large Caribbean landmasses and could disperse
along its continuous coast. A continuous land bridge, so called GAAR-
landia (Greater Antilles-Aves Ridge), connecting the South American
continent with the Greater Antilles and dating to ca. 3335 mya is
thought to have existed (Iturralde-Vinent, 2006). Unfortunately, there is
no reliable substitution rate available for the COI gene of mites. Previous
attempts (Salomone et al., 2002; Heethoff et al., 2007) to infer diver-
gence times in oribatid mites used a general arthropod substitution rate
of 11.15%/MY (DeSalle et al., 1987). However, to unambiguously link
geological events to particular divergence events in Thalassozetes a
reliable substitution rate for oribatid mites is required, as rates might
differ considerably among taxa. Hence, we refrained from applying a
standard arthropod substitution rate to our data and thus cannot relate
the radiation of the cryptic species to any known geological event. But
given the species tree based on all gene fragments and the observed large
interspecic pairwise distances in the COI gene, we can at least state that
a common ancestor split into the Western Caribbean T. balboa that
persisted on the coasts of Central America and into the ancestor of the
cryptic species complex that radiated subsequently in the Eastern
Caribbean, and that the radiation of Caribbean Thalassozetes is not
something very recent. This scenario supports the GAARlandia hy-
pothesis (Iturralde-Vinent, 2006) because this land bridge may have
provided an avenue for the Thalassozetes ancestral species to colonize
the Greater Antilles from South America. The breakup of GAARlandia
resulted in the split between the ancestor of the mainland Caribbean
T. balboa and the island taxa, which further diversied due to the sub-
sequent submergence and emergence of Antillean islands.
4.3. Implications for other taxa and geographic regions
The present case of cryptic intertidal arthropods conrms
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
morphological stasis as an important evolutionary process induced by
the extreme intertidal environment. Therefore, intertidal organisms can
be expected to contain further cryptic species. Especially taxa with poor
dispersal abilities may harbor many cryptic species complexes. In
intertidal mites, there are several cases of poor dispersers with unusually
wide distribution areas spanning a few thousand kilometers. For
example, Schusteria melanomerus occurring from coasts of Kenya to
shores of South Africa (Pngstl, 2016), Fortuynia smiti with records from
New Caledonia and from Singapore (Pngstl, 2015), Fortuynia rotunda
with occurrences in southern Africa and in Japan, or Fortuynia elamellata
being reported from southern Africa, Japan and New Zealand (Pngstl
and Schuster, 2014). These are just a few cases of potential cryptic mite
species and the list is surely longer. The same may apply to other taxa
dwelling in the marine littoral, as for example non-winged/ightless
insects. Moreover, geographic areas with many archipelagos and
oceanic islands separated by vast stretches of open Ocean, such as the
Caribbean or the Southeast Asian Sunda Region, are most likely hiding
large numbers of cryptic species across diverse littoral taxa.
4.4. The problematic nature of dealing with cryptic species
For taxonomists performing faunistic or taxonomic investigations,
cryptic taxa may become an issue, as they will probably remain unde-
tected and the researcher will be left scratching his head about an
intangible ‘intraspecicvariation. Detecting cryptic species usually
requires integrative approaches including multivariate morphometrics,
molecular tools, chemical assays, intensive sampling, crossing etc.
(Skoracka et al., 2015). DNA barcoding initiatives have revealed a
considerably large number of cryptic species in the last few years (e.g.
Hebert et al., 2004; Smith et al., 2006; Vasconcelos et al., 2016; Lavinia
et al., 2017), but only those collaborating with taxonomic specialists
unraveled the complex nature of these cases (e.g. Hebert et al., 2004;
Van Ginneken et al., 2017; Wagner et al., 2021).
However, detecting cryptic species is not enough, formally naming
them is even more crucial for a number of reasons. First, it is the only
way to ensure that scientists are talking about the same taxon, second,
biological data linked to an unnamed species loses value because other
authors cannot easily build on these data, and third, taxa need to be
named for being included in conservation programs (Pante et al., 2015;
c et al., 2017). The latter is of major importance especially for
cryptic species that are endemics occurring on very small islands, as for
example most of the Caribbean Thalassozetes species complex. Slight
changes in these locally restricted environments can have tremendous
impacts on the species (Bickford et al., 2007). Apart from conservation,
unnamed species are also unavailable to biological control and pest
management and the failure to recognize pathogenic cryptic species
might have serious negative consequences (Bickford et al., 2007). Thus,
naming cryptic species is important as it allows to communicate them
with other elds of science that use Linnaean binomials in their research
ser et al. 2017).
Despite these important reasons, many cryptic species remain un-
named (Pante et al., 2015) and somehow get lost in literature as ‘species
B, ‘species 3etc. Researchers usually refrain from formally naming a
species because of a lack of support of species justication, the lack of
knowledge about diagnosing new species using non-morphological
characters, the unwillingness to perform a formal description, the dif-
culties of publishing species descriptions in high impact factor journals
(Pante et al., 2015) and the ongoing controversy about species concepts
and their proper application. But most of these problems can be easily
overcome as shown by the following examples: several authors (Cook
et al., 2010; J¨
orger and Schr¨
odl, 2013) provided specic guidelines for
how to describe and name a cryptic species based on diagnostic DNA
sequence characters only. Others (Wang et al., 2016; Deli´
c et al., 2017)
published exemplary descriptions of cryptic species in high impact fac-
tor journals and hence provided excellent standard works.
In accordance with the above mentioned authors, we propose to take
the following actions when naming a cryptic species: (I) state which
species concept was used to clarify the reasoning of species delimitation,
(II) provide diagnostic characters from different types of data (molecular
genetic markers, morphometric variables, ecological traits, geographic
distributions etc.), (III) provide a depiction of the species and/or of
important morphological features (photograph, drawing, electron
micrograph etc.), (IV) register and upload data to online repositories
(GenBank, ZooBank), (V) deposit holotypes and paratypes in a museum,
(VI) if valid for all cryptic species, provide a clear reference to the
original description of the nominal species or provide own descriptions
as supplementary les and (VII) if possible, provide distribution areas as
allopatric endemics may be identied based on their geographic origin.
However, all these recommendations should not just be seen as a stan-
dard for describing cryptic species, they should apply more generally for
all species descriptions. In this way, species, no matter if cryptic or not,
are treated the same way and named under the same conditions.
To sum up, an integrative taxonomic approach is vital to detect and
understand the phenomenon of cryptic diversity and the association
with a formal species description makes it available for further impor-
tant research, biodiversity estimates and conservation management. A
recent study (Kuroshunova et al., 2019) argued that the cryptic species
concept needs to be reconsidered because with progressing methodology
distinguishing characters will be found rendering the formerly cryptic
species as ‘non-crypticspecies. While this may be true, we think we
should not spend too much time discussing about an eternally valid
denition of cryptic species, we rather should focus on nding and
classifying these diverging taxa and on understanding the evolutionary
mechanisms responsible for the similar phenotypes.
We are grateful to Gabriel de Los Santos (Curator, Museo Nacional de
Historia Natural Prof. Eugenio de Jesús Marcano, Dominican Repub-
lic), Diomedes Quintero (Director, Museo de Invertebrados Fairchild,
Universidad de Panam´
a) and Lil Marie Camacho (Scientic Permits
Ofcer, Smithsonian Tropical Research Institute, Panam´
a) for their help
in administrating the eld trips and in applying for respective permits.
We thank Susan Mahon (Director, McGill Bellairs Research Institute,
Barbados), Mark Vermej (Director, CARMABI Marine Research Station,
Curaçao) and Plinio Gondola (Scientic Coordinator, Bocas Del Toro
Research Station STRI, Panam´
a) and their staff for providing accom-
modation, infrastructure, eldwork permissions and help in every
respect. Thanks also to Clare Morall (St. Georges University, Grenada)
and Justin Rennie (Ministry of Agriculture, Forestry and Fisheries,
Grenada) for organizational help and support. We thank Serge Kreiter
(Montpellier SupAgro, France) for giving us advice concerning our eld
trip to Martinique and Guadeloupe. We are also grateful to the local
Caribbean authorities, especially to the Dominican Republican Minis-
terio de Medio Ambiente y Recursos Naturales and the Vice-Minister of
Areas Protegidas y Biodiversidad, as well as the Panamanian Ministerio
de Ambiente (MiAmbiente) and Director de ´
Areas Protegidas y Vida
Silvestre for issuing important collection and export permits.
This investigation was funded by the Austrian Science Fund (FWF):
[P 28597].
Author Contributions
T.P. performed the sampling, all morphometric measurements and
wrote large parts of the paper, A.L. assisted in the sampling and per-
formed molecular genetic laboratory work, J.B. performed all analyses
based on morphometric data and S.K. analyzed and interpreted molec-
ular genetic results.
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
Appendix A
Information on sampling location and GenBank accession numbers for COI, EF-1
and 18S sequences comprising all specimens included in genetic
GenBank accession nr.
Country Location Sample ID species coordinates COI efa 18S
Curaçao Boca Ascenci´
on CU_15I_2 T. dushi 12.273242, 69.052882 MZ169923
CU_15II_1 MZ169924 MZ220224 MZ220318
CU_15II_2 MZ169925 MZ220225 MZ220317
CU_16_1 12.273342, 69.052667 MZ169926 MZ220226 MZ220316
Dominican Republic Boca Chica DR_04_1 T. dushi 18.44781969.620430 MZ169927 MZ220227 MZ220315
DR_04_2 MZ169928 MZ220228 MZ220314
DR_04_3 MZ220229 MZ220313
Dominican Republic El Lim´
on DR_11II_1 T. samanae 19.32428569.482856 MZ169930 MZ220230
DR_11II_3 MZ169931 MZ220231 MZ220312
Guadeloupe Capesterre-belle-Eau GU_09_03 T. guadeloupensis 16.034611, 61.564938 MZ169968 MZ220261 MZ220294
GU_09_1 MZ169969
GU_09_2n MZ169970 MZ220262 MZ220293
GU_09_3 MZ169971
GU_09_4 MZ169972 MZ220263 MZ220292
GU_09_5 MZ169973 MZ220264 MZ220291
Guadeloupe Sainte-Anne GU_13_1 T. guadeloupensis 16.234413, 61.363318 MZ169974 MZ220265 MZ220290
GU_13_2 MZ169975 MZ220266 MZ220289
Martinique Pointe du Bout MA_02_1 T. martiniquensis 14.55854261.053438 MZ169976 MZ220267 MZ220288
MA_02_2 MZ169977 MZ220268 MZ220287
MA_02_3 MZ169978 MZ220269 MZ220286
Martinique La Trinit´
e MA_08_1 T. martiniquensis 14.74051160.953304 MZ169979
MA_08_2n MZ169980 MZ220270 MZ220285
Grenada Levera Beach GR_05_1 T. grenadensis 12.22818661.613385 MZ169932
GR_05_2 MZ169933 MZ220232 MZ220311
Grenada Levera Beach GR_06_08 T. grenadensis 12.2288861.614432 MZ169934 MZ220233
GR_06_1 MZ169935 MZ220234
GR_06_2 MZ169936 MZ220235 MZ220310
GR_06_3 MZ169937 MZ220236
GR_06_4 MZ169938 MZ220237
GR_06_5 MZ169939 MZ220238 MZ220309
GR_06_6n MZ169940 MZ220239
GR_06_9 MZ169941
Grenada La Sagesse GR_07_1 T. grenadensis 12.02345661.669976 MZ169942 MZ220240
GR_07_2 MZ169943
GR_07_3 MZ169944 MZ220241 MZ220308
Grenada La Sagesse GR_08_2 T. grenadensis 12.02351261.670203 MZ169945 MZ220242 MZ220307
Grenada La Sagesse GR_09_1 T. grenadensis 12.023967, 61.671536 MZ169946
Grenada La Sagesse GR_10_2 T. grenadensis 12.02351261.670203 MZ169947 MZ220243 MZ220306
GR_10_3 MZ169948
GR_10_4 MZ169949 MZ220244
GR_10_5 MZ169950 MZ220245
GR_10_6 MZ169951 MZ220246
Grenada Petite La Sagesse GR_11_1 T. grenadensis 12.01750161.675096 MZ169952 MZ220247 MZ220305
Grenada Petite La Sagesse GR_12_1 T. grenadensis 12.01865961.673232 MZ169953
GR_12_2 MZ169954
GR_12_2a MZ169955 MZ220248 MZ220304
GR_12_3 MZ169956 MZ220249 MZ220303
Grenada Devils Bay GR_13_1 T. grenadensis 12.00665361.796438 MZ169957 MZ220250 MZ220302
GR_13_10 MZ169958 MZ220251 MZ220301
GR_13_2 MZ169959 MZ220252
GR_13_3 MZ169960 MZ220253 MZ220300
GR_13_4 MZ169961 MZ220254 MZ220299
GR_13_5 MZ169962 MZ220255 MZ220298
GR_13_6 MZ169963 MZ220256 MZ220297
GR_13_7 MZ169964 MZ220257 MZ220296
GR_13_8 MZ169965 MZ220258 MZ220295
GR_13_9 MZ169966 MZ220259
Grenada Pink Gin Beach GR_14_1 T. grenadensis 12.008939, 61.791091 MZ169967 MZ220260
a Isla Col´
on T_PA_35_1 T. balboa 9.36289882.239319 MZ170018
T_PA_35_2 MZ170019
a Isla Col´
on T_PA_37_1 T. balboa 9.37082182.239908 MZ170020
T_PA_37_10 MZ170021
T_PA_37_2 MZ170022
T_PA_37_3 MZ170023 MZ220277 MK035018
T_PA_37_5 MZ170024
T_PA_37_6 MZ170025
(continued on next page)
T. Pngstl et al.
Molecular Phylogenetics and Evolution 163 (2021) 107240
GenBank accession nr.
Country Location Sample ID species coordinates COI efa 18S
T_PA_37_8 MZ170026
T_PA_37_9 MZ170027 MZ220278 MZ220279
a Isla Col´
on T_PA_39_1 T. balboa 9.38545482.23524 MZ170028
T_PA_39_2 MZ170029
T_PA_39_3 MZ170030
T_PA_39_4 MZ170031
T_PA_39_5 MZ170032
T_PA_39_6 MZ170033
a Isla Col´
on T_PA_43_1 T. balboa 9.41505782.330787 MZ170034
T_PA_43_2 MZ170035
T_PA_43_3 MZ170036 MK035019
T_PA_43_4 MZ170037
T_PA_43_5 MZ170038
Florida Key Biscayne T_FL_03_1 T. balboa 25.67729680.164818 MZ170014
T_FL_03_2 MZ170015
T_FL_03_3 MZ170016
T_FL_03_4 MZ170017
Bahamas Paradise Island T_BH_03_1 T. paradisi 25.085983, 77.29966 MZ170003
Bahamas Compass Point T_BH_10_1 T. paradisi 25.065252, 77.470981 MZ170004
T_BH_10_2 MZ170005
T_BH_10_3 MZ170006 MZ220275 MZ220281
T_BH_10_4 MZ170007
T_BH_10_5 MZ170008
T_BH_10_6 MZ170009
T_BH_10_7 MZ170010
T_BH_10_8 MZ170011 MZ220276 MZ220280
Bahamas Paradise Island T_BH_25_1 T. paradisi 25.086345, 77.301111 MZ170012
T_BH_25_2 MZ170013
Barbados Bathsheba T_BA_13_1 T. barbara 13.213011, 59.520318 MZ169981
Barbados Bathsheba T_BA_14_1 T. barbara 13.213834, 59.521433 MZ169982 MZ220271 MZ220284
Barbados Bathsheba T_BA_15_1 T. barbara 13.21393, 59.521825 MZ169983 MZ220272 MZ220283
Barbados Bridgetown T_BA_19_1 T. barbara 13.078423, 59.612556 MZ169984
T_BA_19_2 T. grenadensis MZ169985
T_BA_19_3 MZ169986
T_BA_19_4 MZ169987
T_BA_19_5 MZ169988
T_BA_19_6 MZ169989
Barbados St. Peters Bay T_BA_20_1 T. barbara 13.240601, 59.645153 MZ169990
T_BA_20_2 13.240601, 59.645153 MZ169991
Barbados St. Peters Bay T_BA_21_1 T. barbara 13.240219, 59.645069 MZ169992
Barbados Oistins T_BA_22_1 T. barbara 13.062537, 59.541903 MZ169993
Barbados Miami Beach T_BA_24_1 T. grenadensis 13.06055959.540786 MZ169994
Barbados Miami Beach T_BA_25_1 T. barbara 13.06000659.538893 MZ169995
Barbados Bathsheba T_BA_28_1 T. barbara 13.212719, 59.517116 MZ169996
Barbados Bathsheba T_BA_29_1 T. barbara 13.212719, 59.517116 MZ169997 MZ220273 MZ220282
T_BA_29_2 MZ169998
Barbados Bathsheba T_BA_30_1 T. barbara 13.212719, 59.517116 MZ169999 MK035020
T_BA_30_2 MW289085
MZ220274 MW298484
T_BA_30_3 MZ170000
T_BA_30_4 T. barbara MZ170001
Barbados Bathsheba T_BA_31_1 T. barbara 13.216753, 59.526811 MZ170002
Sequence from Pngstl et al. (2021);
Sequence published by Pngstl et al. (2019b).
Appendix B. Supplementary material
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... Carinozetes bermudensis and Carinozetes mangrovi, both species originally described from Bermuda, just like F. atlantica, were found to be widespread on Caribbean coasts whereas a molecular genetic investigation revealed them to consist of five distinct genetic lineages, probably representing different species. Furthermore, the allegedly widespread Thalassozetes barbara [6], another intertidal mite species, was shown to consist of at least seven different species with nearly each representing a Caribbean island endemic [14]. ...
... The Caribbean intertidal Carinozetes mangrovi and Carinozetes bermudensis, for example, show wide distributions but consist of five distinct genetic lineages that cannot be distinguished based on morphology [13]. Another recent study [14] revealed the formerly widespread Caribbean intertidal mite Thalassozetes barbara to consist of seven morphologically identical but genetically distinct species whereas nearly all species represent island endemics. ...
... Moreover, all individuals clustered congruently in the two single gene trees. In the cryptic Caribbean Thalassozetes species, on the other hand, the 18S marker was not useful to separate the taxa with confidence [14] and even in the clear morphospecies Fortuynia churaumi and F. shibai from Japan this was not possible [48] due to incomplete lineage sorting. The two herein used markers thus clearly identify the Fortuynia from Barbados as a distinct lineage that is reproductively isolated from the other investigated populations. ...
Full-text available
A molecular genetic and morphometric investigation revealed the supposedly widespread Caribbean and Western Atlantic intertidal oribatid mite species Fortuynia atlantica to comprise at least two different species. Although there are no distinct morphological differences separating these taxa, COI and 18S sequence divergence data, as well as different species delimitation analyses, clearly identify the two species. Fortuynia atlantica is distributed in the northern Caribbean and the Western Atlantic and the new Fortuynia antillea sp. nov. is presently endemic to Barbados. Vicariance is supposed to be responsible for their genetic diversification and stabilizing selection caused by the extreme intertidal environment is suggested to be the reason for the found morphological stasis. The genetic structure of Fortuy-nia atlantica indicates that Bermudian populations are derived from the northern Caribbean and thus support the theory of dispersal by drifting on the Gulf Stream. Haplotype network data suggest that Bermudian and Bahamian populations were largely shaped by coloniza-tion, expansion and extinction events caused by dramatic sea level changes during the Pleistocene. A preliminary phylogenetic analysis based on 18S gene sequences indicates that the globally distributed genus Fortuynia may be a monophyletic group, whereas Caribbean and Western Atlantic members are distinctly separated from the Indo-Pacific and Western Pacific species.
... Adaptation to homogeneous and highly constraining environments, such as soil, has been suggested to impose stabilising selection for morphology, resulting in highly conserved morphological traits within evolutionary lineages (Colborn et al., 2001;Lefébure et al., 2006;Pfingstl et al., 2021). Such selection complicates the use of diagnostic morphological characters for classical taxonomic delimitation of species, something that has been identified as resulting in broader species diversity being lumped within a more limited number of taxonomic names (e.g. ...
... Such selection complicates the use of diagnostic morphological characters for classical taxonomic delimitation of species, something that has been identified as resulting in broader species diversity being lumped within a more limited number of taxonomic names (e.g. Cicconardi et al., 2013;Morek et al., 2021;Pfingstl et al., 2021). ...
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Specialisation to the soil environment is expected to constrain the spatial scale of diversification within animal lineages. In this context, flightless arthropod lineages, adapted to soil environments, but with broad geographical ranges, represent something of an anomaly. Here we investigate the diversification process within one such ‘anomalous’ soil specialist, an eyeless and flightless beetle species strongly adapted to the endogean environment but distributed across several oceanic islands. Canary Islands. Geomitopsis franzi Coiffait, 1978 (Coleoptera, Staphylinidae). We performed an integrative study, including molecular phylogenetics, population genomics and morphometry. Four DNA regions (two mitochondrial and two nuclear) were amplified and sequenced for 159 specimens from 58 localities sampled across five islands for phylogenetic analyses, and a dated phylogenetic tree was obtained using a mitogenome dataset. ddRAD‐seq data were generated to evaluate mtDNA lineages in sympatry against the biological species concept. We found high levels of genetic differentiation (>8% COI gene divergence) among populations from different islands and among geographically coherent lineages within single islands. Lineages within Tenerife presented significant patterns of isolation by distance, with ddRAD‐seq providing evidence that lineages represent biological species. Morphometric analyses revealed limited variation. Geomitopsis franzi is comprised of at least seven lineages that merit consideration as biological species, and is best considered as a complex of cryptic species. The limited morphological variation across these lineages is consistent with adaptation to the endogean environment placing strong constraints on morphological change. The evolution of cryptic species should be favoured when such constraints are coupled with limited dispersal ability, a trait that broadly characterises the soil mesofauna.
... In recent studies of species delimitation based on single-locus data, researchers were always using both tree-and distance-based methods to make the results more persuasive (Blair & Bryson 2017;Pfingstl et al. 2021). Therefore, three different approaches were used for the species delimitation in this work: (1) General Mixed Yule Coalescent model (GMYC) (Fujisawa & Barraclough 2013), tree-based method; (2) Poisson Tree Process (PTP) (Zhang et al. 2013), tree-based method; (3) Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2012), distance-based method. ...
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Epeorus Eaton, 1881 is a diverse mayfly genus in Heptageniidae comprising more than 100 species which are further divided into nine subgenera and several species groups. However, the classification and the phylogenetic relationships among them are still uncertain. Here, 15 complete mitochondrial genomes of Epeorus were sequenced and compared together with six available ones of same genus in the NCBI database. Based on morphological classification, the 21 mitogenomes were classified into six subgenera (Proepeorus, Epeorus s.str., Belovius, Iron, Caucasiron and Siniron) and four species groups (G1, G2, montanus and longimanus). Among all analyzed mitogenomes, the gene rearrangement of trnI-trnM-trnQ-NCR-ND2 was first found occurring in three species of group G1, whereas the gene block trnI-trnM-trnQ-trnM-ND2 was observed in all other mitogenomes of Epeorus. Furthermore, the genetic composition and codon usage of species in group G1 were also significantly different from all other Epeorus species, except group longimanus. The intergenic spacer between trnA and trnR, which has the stem-loop secondary structure, occurred in all 21 mitogenomes, and the sequences of stems and loops were conserved within species groups. Furthermore, the phylogenetic analyses strongly support the monophyly of all species groups, although three of six recognized subgenera Proepeorus, Belovius, and Iron, were shown as the non-monophyletic groups.
... orbicularis have been shown to be close genetic groups [6] and could represent recently diverged species that have not accumulated any morphological differences yet. However, the nine species sampled in the present study are part of three very divergent genera (i.e., Fuscifolium, Porphyra, and Pyropia; [68]), and the evolutionary convergence or morphological stasis linked to life in the highly stressful intertidal zone (e.g., [71]) could be hypothesized in bladed Bangiales. ...
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Morphologically similar but genetically distinct species have been termed cryptic and most have been assumed to be ecologically similar. However, if these species co-occur at a certain spatial scale, some niche differences at finer scales should be expected to allow for coexistence. Here, we demonstrate the existence of a disjointed distribution of cryptic bladed Bangiales along spatial (intertidal elevations) and temporal (seasons) environmental gradients. Bladed Bangiales were identified and quantified across four intertidal elevations and four seasons for one year, at five rocky intertidal sites (between 39° S and 43° S) in southern Chile. Species determination was based on partial sequences of the mitochondrial cytochrome c oxidase 1 (COI) gene amplification. To assess species gross morphology, thallus shape, color, and maximum length and width were recorded. Hundreds of organisms were classified into nine Bangiales species belonging to three genera (i.e., Fuscifolium, Porphyra, and Pyropia), including five frequent (>97% of specimens) and four infrequent species. All species, except for Pyropia saldanhae, had been previously reported along the coasts of Chile. The thallus shape and color were very variable, and a large overlap of the maximum width and length supported the cryptic status of these species. Multivariate analyses showed that the main variable affecting species composition was intertidal elevation. Species such as Py. orbicularis were more abundant in low and mid intertidal zones, while others, such as Po. mumfordii and Po. sp. FIH, were principally observed in high and spray elevations. Despite all numerically dominant species being present all year long, a slight effect of seasonal variation on species composition was also detected. These results strongly support the existence of spatial niche partitioning in cryptic Bangiales along the Chilean rocky intertidal zone.
... In recent studies of species delimitation based on single-locus data, researchers were always using both tree-and distance-based methods to make the results more persuasive (Blair & Bryson 2017;Pfingstl et al. 2021). Therefore, three different approaches were used for the species delimitation in this work: (1) General Mixed Yule Coalescent model (GMYC) (Fujisawa & Barraclough 2013), tree-based method; (2) Poisson Tree Process (PTP) (Zhang et al. 2013), tree-based method; (3) Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2012), distance-based method. ...
A new subgenus, Siniron subgen. n. , is established for five Chinese species of Epeorus Eaton, 1881 to recognize their distinct difference from other subgenera: 1) in nymphs, tergalius I widely expanded anteriorly while tergalius VII curved but unfolded, well developed paired spines on abdominal terga; 2) in adults, unique coloration of wings, penis with distinct median titillators. Among them, nymphal stages of four previously known species, E. ( S .) sinensis (Ulmer, 1925), E. ( S .) dayongensis Gui & Zhang, 1992, E. ( S .) herklotsi (Hsu, 1936b) and E. ( S. ) ngi Gui, Zhou & Su, 1999, are described for the first time and imaginal stages are also re-described. The fifth species, which has apically pigmented hind wings in imago and protuberances on pronotum in nymph, is described as a new species E. ( S. ) tuberculatus sp. n. All these species can also be delimited by COI sequences. In addition, their distribution in China is provided.
... From the evolutionary emergence of primitive organisms to today's broad variety of organisms, people have been constantly exploring how many species there are on the earth and what kind of evolutionary relationship among species. With the development of open science and technological innovation, methods of species identification range from using morphological characteristics to the integration of various methods (e.g., molecular biology, bioinformatics, bionomics) [1][2][3][4][5], which help us gain a more in-depth understanding of the evolutionary process between organisms and their accurate position in the tree of life. Due to the multi-disciplines combination and the improvement of sharing databases, many misclassifications hidden in the past have been gradually discovered, and their key morphological boundaries have also been rewritten. ...
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With the development of open science and technological innovation, using sharing data and molecular biology techniques in the study of taxonomy and systematics have become a crucial component of plants, which undoubtedly helps us discover more hidden outliers or deal with difficult taxa. In this paper, we take Dennstaedtia smithii as an example, based on sharing molecular database, virtual herbarium and plant photo bank, to clarify the outliers that have been hidden in Dennstaedtia and find the key morphological traits with consistent of molecular systematics. In molecular phylogenetic analyses, we used rbcL, rps4, psbA-trnH and trnL-F sequences from 5 new and 49 shared data; the results showed that Dennstaedtia smithii is nested within Microlepia rather than Dennstaedtia. We further studied the morphological characters based on the phylogeny result and found that D. smithii is distinguished from other species of Dennstaedtia by spore ornamentation and the unconnected of grooves between rachis and pinna rachis. According to morphological and molecular phylogenetic studies, our results supported that D. smithii should be a new member of Microlepia and renamed Microlepia smithii (Hook.) Y.H. Yan. Finding hidden outliers can promote the consistency of morphological and molecular phylogenetic results, and making the systematic classification more natural.
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Island biogeographers have long sought to elucidate the mechanisms behind biodiversity genesis. The Caribbean presents a unique stage on which to analyze the diversification process, due to the geologic diversity among the islands and the rich biotic diversity with high levels of island endemism. The colonization of such islands may reflect geologic heterogeneity through vicariant processes and/ or involve long-distance overwater dispersal. Here, we explore the phylogeography of the Caribbean and proximal mainland spiny orbweavers (Micrathena, Araneae), an American spider lineage that is the most diverse in the tropics and is found throughout the Caribbean. We specifically test whether the vicariant colonization via the contested GAARlandia landbridge (putatively emergent 33–35 mya), long-distance dispersal (LDD), or both processes best explain the modern Micrathena distribution. We reconstruct the phylogeny and test biogeographic hypotheses using a ‘target gene approach’ with three molecular markers (CO1, ITS-2, and 16S rRNA). Phylogenetic analyses support the monophyly of the genus but reject the monophyly of Caribbean Micrathena. Biogeographical analyses support five independent colonizations of the region via multiple overwater dispersal events, primarily from North/Central America, although the genus is South American in origin. There is no evidence for dispersal to the Greater Antilles during the timespan of GAARlandia. Our phylogeny implies greater species richness in the Caribbean than previously known, with two putative species of M. forcipata that are each single-island endemics, as well as deep divergences between the Mexican and Floridian M. sagittata. Micrathena is an unusual lineage among arachnids, having colonized the Caribbean multiple times via overwater dispersal after the submergence of GAARlandia. On the other hand, single-island endemism and undiscovered diversity are nearly universal among all but the most dispersal-prone arachnid groups in the Caribbean.
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A challenge for taxonomists all over the world and across all taxonomic groups is recognizing and delimiting species, and cryptic species are even more challenging. However, an accurate identification is fundamental for all biological studies from ecology to conversation biology. We used a multidisciplinary approach including genetics as well as morphological and ecological data to assess if an easily recognizable, widely distributed and euryoecious mite taxon represents one and the same species. According to phylogenetic (based on mitochondrial and nuclear genes) and species delimitation analyses, five distinct putative species were detected and supported by high genetic distances. These genetic lineages correlate well with ecological data, and each species could be associated to its own (micro)habitat. Subsequently, slight morphological differences were found and provide additional evidence that five different species occur in Central and Southern Europe. The minuteness and the characteristic habitus of Caleremaeus monilipes tempted to neglect potential higher species diversity. This problem might concern several other “well-known” euryoecious microarthropods. Five new species of the genus Caleremaeus are described, namely Caleremaeus mentobellus sp. nov., C. lignophilus sp. nov., C. alpinus sp. nov., C. elevatus sp. nov., and C. hispanicus sp. nov. Additionally, a morphological evaluation of C. monilipes is presented.
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A comprehensive study of the intertidal oribatid mite fauna of southern Japanese islands revealed the presence of the selenoribatid Arotrobates granulatus Luxton, 1992 and two yet undescribed species. The latter are herein described as Indopacifica taiyo n. sp., occurring from the Southern to the Central Ryukyus, and Indopacifica tyida n. sp., which was only found on the most western island of the Ryukyus, namely Yonaguni. A concomitant molecular genetic study using mitochondrial COI and 18S rRNA gene sequences, demonstrated that the phylogeographic pattern of I. taiyo n. sp. reflects recent expansion on the Southern and Central Ryukyus, probably due to existing land bridges during the late Pleistocene. Arotrobates granulatus, on the other hand, shows three distinct lineages, one on Japanese mainland, another on the island of Amami, and the third on part of the Central and Southern Ryukyus. These lineages are most likely the result of the break-up of a large peninsula reaching from China to the Northern Ryukyus about 1.2–1.7 million years ago. Despite emerging land bridges in the late Pleistocene, this species was not able to expand its range again which indicates very low dispersal abilities. Morphometric data of I. taiyo n. sp. show considerable intraspecific variation between island populations correlating with geography. This found variation is suggested to be a result of phenotypic plasticity caused by diverging local environmental factors. From an ecological perspective, all three found species are classified as intertidal rock-dwellers feeding on diverse algae, whereas I. taiyo n. sp. and Arotrobates granulatus occasionally occur in mangrove habitats.
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Background Snow scorpionflies (genus Boreus ) belong to a family of Mecoptera, Boreidae, that has been vastly neglected by entomological researchers due to their shift in seasonality to the winter months. Their activity during this time is regarded as a strategy for predator avoidance and regular sightings on snow fields suggest that this also facilitates dispersal. However, many aspects about snow scorpionflies, especially systematics, taxonomy, distribution of species, phylogenetics and phylogeography have remained fairly unexplored until today. In this study, we fill some of these gaps by generating a reference DNA barcode database for Austrian snow scorpionflies in the frame of the Austrian Barcode of Life initiative and by characterising morphological diversity in the study region. Methods Initial species assignment of all 67 specimens was based on male morphological characters previously reported to differ between Boreus species and, for females, the shape of the ovipositor. DNA barcoding of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene was carried out for all 67 samples and served as a basis for BIN assignment, genetic distance calculations, as well as alternative species delimitation analyses (ABGD, GMYC, bGMYC, bPTP) and a statistical parsimony network to infer phylogenetic relationships among individual samples/sampling sites. Results Morphological investigations suggested the presence of both Boreus hyemalis and Boreus westwoodi in Austria. DNA barcoding also separated the two species, but resulted in several divergent clades, the paraphyly of B. westwoodi in Austria, and high levels of phylogeographic structure on a small geographic scale. Even though the different molecular species delimitation methods disagreed on the exact number of species, they unequivocally suggested the presence of more than the traditionally recognized two Boreus species in Austria, thus indicating potential cryptic species within the genus Boreus in general and especially in B. westwoodi .
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A decade ago the Caribbean was almost completely uncharted in terms of intertidal ameronothroid mites. Now the present data show that these organisms are a common component of the fauna of Caribbean shorelines. Two families of Ameronothroidea are present, the Fortuyniidae with three genera and four species and the Selenoribatidae with five genera and nine species. The most common species are the fortuyniid Alismobates inexpectatus and the selenoribatid Carinozetes mangrovi, both taxa were found in the Northern Caribbean, the Greater and Lesser Antilles as well as on Central American coasts. Six species are endemic to the Caribbean, Litoribates bonairensis, L. floridae, Schusteria marina, Thalassozetes balboa, T. barbara and Thasecazetes falcidactylus. Biogeographic patterns suggest that the genera Carinozetes and Litoribates may have evolved and diversified in the Caribbean region and that the Western Atlantic Bermudian intertidal oribatid mite fauna was largely shaped by Caribbean colonizers. Most of the species found in the Caribbean are typical rock dwellers and only a minority is represented by exclusive mangrove specialists. These species are seriously threatened by the significant progressive decline of mangrove ecosystems throughout the Caribbean.
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1. raxmlGUI is a graphical user interface to RAxML, one of the most popular and widely used softwares for phylogenetic inference using maximum likelihood. 2. Here we present raxmlGUI 2.0, a complete rewrite of the GUI which seamlessly integrates RAxML binaries for all major operating systems with an intuitive graph-ical front-end to setup and run phylogenetic analyses. 3. Our program offers automated pipelines for analyses that require multiple successive calls of RAxML, built-in functions to concatenate alignment files while automatically specifying the appropriate partition settings, and one-click model testing to select the best substitution models using ModelTest-NG. In addition to RAxML 8.x, raxmlGUI 2.0 also supports the new RAxML-NG, which provides new functionality and higher performance on large datasets. 4. raxmlGUI 2.0 facilitates phylogenetic analyses by coupling an intuitive interface with the unmatched performance of RAxML. K E Y W O R D S bioinformatics, evolutionary biology, molecular biology, phylogenetics, software
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The clingfish (Gobiesocidae) genus Gouania (Nardo 1833) is endemic to the Mediterranean Sea and inhabits, unlike any other vertebrate species in Europe, the harsh intertidal environment of gravel beaches. Following up on a previous phylogenetic study, we revise the diversity and taxonomy of this genus, by analysing a comprehensive set of morphological (meristics, morphometrics, micro computed tomography imaging), geographical and genetic (DNA‐barcoding) data. We provide descriptions of three new species, G. adriatica sp. nov., G. orientalis sp. nov., G. hofrichteri sp. nov. as well as re‐descriptions of G. willdenowi (Risso 1810) and G. pigra (Nardo 1827) and assign neotypes for the latter two species. In addition to elucidating the complex taxonomic situation of Gouania, we discuss the potential of this enigmatic clingfish genus for further ecological, evolutionary and biodiversity studies that might unravel even more diversity in this unique Mediterranean fish radiation.
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Bark beetles are feared as pests in forestry but they also support a large number of other taxa that exploit the beetles and their galleries. Among arthropods, mites are the largest taxon associated with bark beetles. Many of these mites are phoretic and often involved in complex interactions with the beetles and other organisms. Within the oribatid mite family Scheloribatidae, only two of the three nominal species of Paraleius have been frequently found in galleries of bark beetles and on the beetles themselves. One of the species, P. leontonychus, has a wide distribution range spanning over three ecozones of the world and is believed to be a host generalist, reported from numerous bark beetle and tree species. In the present study, phylogenetic analyses of one mitochondrial and two nuclear genes identified six well supported, fairly divergent clades within P. leontonychus which we consider to represent distinct species based on molecular species delimitation methods and largely congruent clustering in mitochondrial and nuclear gene trees. These species do not tend to be strictly host specific and might occur syntopically. Moreover, mito-nuclear discordance indicates a case of past hybridization/introgression among distinct Paraleius species, the first case of interspecific hybridization reported in mites other than ticks.
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Chamobates borealis (Trägårdh 1902) has been considered by some authors as a junior synonym of Chamobates pusillus (Berlese 1895). In this study we used an integrated taxonomy approach, comparing mitochondrial coding gene COI and morphological ontogeny of these species to clarify their systematic status. The Bayesian inference tree based on COI sequences of C. borealis and C. pusillus, as well as C. birulai (Kulczyński 1902), C. bispinosus Mahunka, 1987, C. cuspidatus (Michael 1884) and C. rastratus (Hull 1914) separated all these species. In terms of the morphology, the adults of C. borealis and C. pusillus have similar body size and shape, thin aggenital setae and two lateral teeth on the rostrum, but C. borealis has the medial incision between these teeth, which is absent in C. pusillus. The adults of these species differ also from each other by the shape of bothridial setae, size of area porose Aa, location of seta lm and lyrifissure im, and the shape of most setae on the hysterosoma. The morphological ontogeny of these species is similar, but the larva and nymphs of C. borealis differ from those of C. pusillus by the length of some prodorsal and gastronotal setae, and the nymphs of C. borealis have a humeral organ, which is absent in C. pusillus. The presence of a humeral organ in some Chamobates species supports a clade inferred by COI sequence data.