Evolution and biogeography of the Haploniscus belyaevi species complex (Isopoda: Haploniscidae) revealed by means of integrative taxonomy

Abstract

The role of geomorphological features as drivers for benthic deep-sea biodiversity remains poorly understood. By disentangling the putative Haploniscus belyaevi Birstein, 1963a species complex from the abysso-hadal Kuril-Kamchatka Trench (KKT) region in the North-west Pacific Ocean, we aim to shed light on deep-sea differentiation and how it is related to potential bathymetric barriers such as the KKT and the Kuril-Island Ridge (KIR). Our integrative taxonomic approach featured morphological and molecular delimitation methods, also considering the post-marsupial development due to pronounced sexual dimorphism. Mitochondrial 16S and COI markers were sequenced and several molecular species delimitation methods were applied. By combining the different results we were able to delineate six distinct species within the belyaevi complex, including several morphologically cryptic species, and found hints of three additional species groups in the complex. Even though several of these species were distributed across the KKT and/or KIR, limited gene flow and depth-differentiation were indicated supporting previous notions that these geomorphological features play a role in deep-sea benthos speciation.

Full text

Research Article
Evolution and biogeography of the Haploniscus belyaevi species
complex (Isopoda: Haploniscidae) revealed by means of
integrative taxonomy
HENRY KNAUBER
1,2
, JONA R. SILBERBERG
1,2
, ANGELIKA BRANDT
1,2
& TORBEN RIEHL
1,2
1
Department of Marine Zoology, Senckenberg Research Institute and Natural History Museum, Section Crustacea,
Senckenberganlage 25, Frankfurt, 60325, Germany
2
Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Johann Wolfgang Goethe University Frankfurt,
Max-von-Laue-Str. 13, Frankfurt, 60438, Germany
(Received 12 October 2021; accepted 4 July 2022)
The role of geomorphological features as drivers for benthic deep-sea biodiversity remains poorly understood. By
disentangling the putative Haploniscus belyaevi Birstein, 1963a species complex from the abysso-hadal Kuril-
Kamchatka Trench (KKT) region in the North-west Pacific Ocean, we aim to shed light on deep-sea differentiation and
how it is related to potential bathymetric barriers such as the KKT and the Kuril-Island Ridge (KIR). Our integrative
taxonomic approach featured morphological and molecular delimitation methods, also considering the post-marsupial
development due to pronounced sexual dimorphism. Mitochondrial 16S and COI markers were sequenced and several
molecular species delimitation methods were applied. By combining the different results we were able to delineate six
distinct species within the belyaevi complex, including several morphologically cryptic species, and found hints of three
additional species groups in the complex. Even though several of these species were distributed across the KKT and/or
KIR, limited gene flow and depth-differentiation were indicated supporting previous notions that these geomorphological
features play a role in deep-sea benthos speciation.
Key words: Haploniscidae, Haploniscus, Isopoda, Kuril-Island Ridge
Introduction
The deep-sea benthos supports a remarkable diversity of
invertebrates, with isopod crustaceans being a particularly
species-rich and abundant taxon (e.g., Brandt et al., 2019;
Brix et al., 2018). To date, the underlying factors that
have historically shaped this diversity and the role of
important geomorphological features such as trenches and
ridges are not well understood. Studying the phylogeogra-
phy of deep-sea taxa is hampered by the availability of
few datasets of sufficient size to allow an appropriate
study design in terms of geographic coverage and number
of conspecifics (Taylor & Roterman, 2017). In addition,
taxonomic uncertainty is a major problem when it comes
to deep-sea specimens, as most deep-sea species are not
taxonomically described (Mora et al., 2011) and formally
recognized species often turn out to actually be
complexes of several previously overlooked species when
studied genetically (e.g., Brix et al., 2015;Dan

ıelsd
ottir
et al., 2008; Goffredi et al., 2003).
Species complexes pose a challenge for the biogeog-
raphy and the estimation of marine biodiversity. Often
entailing morphologically highly similar or even cryptic
species they may cause biodiversity underestimation in the
context of classical evaluations of species richness or even-
ness (Vrijenhoek, 2009). Delimiting marine species com-
plexes can be further complicated by phenotypic plasticity,
for example in the form of complex lifestyles or sexual
dimorphism (e.g., Riehl et al., 2012; Vrijenhoek, 2009).
Because species serve as evolutionary units, accurate spe-
cies delimitation is essential as a basis of any phylogeo-
graphic study (Pante et al., 2015). However, species
complexes also bear the potential to unravel deep-sea evo-
lutionary mechanisms (Knowlton, 1993;Mayr,1963).
Phylogeographic patterns of species complexes can provide
valuable insights into how environmental factors, such as
Correspondence to: Torben Riehl. E-mail:
torben.riehl@senckenberg.de
ISSN 1477-2000 print / 1478-0933 online
#2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
https://dx.doi.org/10.1080/14772000.2022.2099477
Systematics and Biodiversity (2022), 20(1): 2099477
Published online 30 Aug 2022

depth and geomorphology, have impacts on deep-sea spe-
ciation (Silva et al., 2021). Herein lies a great potential
and significant opportunity to improve our understanding
of evolution in the deep oceans and, thus, the origins of
deep-sea biodiversity (Taylor & Roterman, 2017).
Since 2012, a series of three expeditions to the deep
North-west Pacific (NWP) have surveyed the fauna of the
wider Kuril-Kamchatka Trench (KKT) region, including
the NWP Abyssal Plain, the KKT itself, the Kuril Basin
of the Sea of Okhotsk (SO), and the bathyal Bussol Strait
and adjacent slope regions of the Kuril-Island Ridge
(KIR) (Brandt et al., 2020). The objectives of these cam-
paigns included the inventory of the deep-sea benthic
fauna and the investigation of potential barrier effects of
the large-scale bathymetric features of the KIR and KKT
using a standardized gear set (Brandt et al., 2020).
Among the samples from the NWP, a species complex
of Haploniscidae Hansen, 1916 (Crustacea: Isopoda) has
been identified (Johannsen et al., 2020) comprising
Haploniscus belyaevi Birstein, 1963a and five additional
morphologically highly similar groups. The proposed H.
belyaevi species complex (henceforward called belyaevi
complex) is particularly promising to disentangle the
influence of environmental factors on its phylogeography
because it was highly abundant in the samples and
widely distributed in the greater KKT region (Johannsen
et al., 2020). It occurred from the SO in the north to the
North-west Pacific Abyssal Plain in the south, across a
subduction zone with a complex seafloor geomorphology
(including trench and ridge) and three biogeographic
provinces (see Watling et al., 2013). Within this geo-
graphic range the belyaevi complexoccurredinsamples
from depths covering the abyssal and hadal zones of the
benthos (see Johannsen et al., 2020)renderingthe
belyaevi complex a particularly good model taxon to
study deep-sea diversification. Diagnostic (morphological/
genetic/genomic) differences between sister species
occurring in neighbouring biogeographic provinces com-
monly indicate interspecific concordance between bio-
geography and phylogeography; in cases where
individual species are distributed across two or more bio-
geographic provinces –as in the case of the belyaevi
complex –shifts in genotype frequencies or significant
levels of differentiation often align with biogeographic
boundaries, providing intraspecific concordance between
biogeography and phylogeography (Bowen et al., 2016).
Likewise, depth-dependent inter-species divergence and
intraspecific differentiation have been frequently observed
in deep-sea taxa (e.g., Havermans et al., 2013; Jennings
et al., 2013;Riehletal.,2018), pointing towards depth as
a major evolutionary factor (see also McCallum & Riehl,
2020). Hence, species complexes occurring in border
regions of biogeographic provinces and at various depths,
such as the belyaevi complex, are of major interest to
investigate patterns of differentiation and to pinpoint the
factors responsible for limiting gene flow.
The belyaevi complex furthermore represents a suitable
model taxon because as benthic brooders and without
adaptations to long-distance dispersal (Brix et al., 2020),
haploniscids should be especially substrate-bound and,
thus, their dispersal and gene flow should be affected by
habitat patchiness and discontinuity (see S. Bober et al.,
2018). With diversity-regulating processes differing at
local, regional, and global scales, the impact of these
processes on the marine fauna varies depending on their
dispersibility (Bowen et al., 2016;Brandtetal.,2004). In
places with strong bathymetric gradients and varying
slope angles, such as the wider KKT region, sediment
coverage varies significantly (e.g., Niedzielski et al.,
2013; Riehl et al., 2020). This can result in a mosaic-like
pattern of sedimented (soft substrate) and rocky (hard sub-
strate) habitats, each with its specialized benthic fauna.
Such interruptions of suitable habitats should furthermore
reduce dispersal in sediment-dwelling organisms, such as
haploniscids, and may therefore promote population dif-
ferentiation in these organisms (Riehl et al., 2020).
The primary goal of this work was to corroborate the
hypothesis that Haploniscus belyaevi sensu Johannsen et al.
(2020) comprises a complex of multiple, closely related
species. This was accomplished by applying the taxonomic
feedback loop, first independently scrutinizing morpho-
logical ontogenetic development, molecular phylogenetics,
biogeography and DNA-based species delimitation and
then synthesizing the results. The species delimitation rep-
resents the essential first step to disentangle the effects of
depth and geomorphological barriers on diversification.
Then, by relating biogeographic with phylogenetic pat-
terns, the second goal was to study historic differentiation
across biogeographic borders, depth strata and large geo-
morphological features to infer their potential effects on
benthic speciation. It was expected that across these afore-
mentioned barriers both intraspecific differentiation as
well as interspecific divergence would become apparent.
Providing a first indication that these factors indeed pro-
mote speciation in the belyaevi complex, this study opens
up new possibilities for future population-genetic and phy-
logeographic investigations on the NWP deep-sea haplo-
niscids and provides the foundation for proper taxonomic
descriptions of the species of the belyaevi complex.
Materials and methods
Study area, sampling handling, and
specimen treatment
All samples analysed in this study were collected in the
greater Kuril-Kamchatka Trench (KKT) region of the
2 H. Knauber et al.

North-west Pacific Ocean (NWP) and its marginal Sea
of Okhotsk (SO). To the north-west of the KKT, the
KIR separates the NWP from the adjacent SO and its
Kuril Basin (3,374 m max. depth). However, several
bathyal straits, of which the Bussol Strait (2,350 m sill
depth) and the Krusenstern Strait (1,920 m sill depth)
are the deepest, allow for exchange of water and poten-
tially also deep-sea fauna between the KKT and the SO
(Malyutina et al., 2018; Tyler, 2002). Both the KKT
and the KIR might represent barriers hard to overcome
for deep-sea species (e.g., J. Bober et al., 2019;
Johannsen et al., 2020) as they also divide the KKT
region into the following biogeographic provinces as
defined by Watling et al. (2013): (i) the Aleutian-Japan
hadal province, restricted to the KKT; (ii) the Northern
Pacific Boreal province, represented by the SO; and (iii)
the North Pacific province, comprising vast NWP abys-
sal plains. Furthermore, for a preliminary biogeographic
analysis, the greater KKT region has been divided into
four geographic zones (SO ¼Sea of Okhotsk,
WTA ¼West Trench Abyssal, CTH ¼Central Trench
Hadal, ETA ¼East Trench Abyssal) based on the bio-
geographic provinces defined by Watling et al. (2013)
but with further adjustments proposed by Johannsen
et al. (2020) to include local bathymetric characteristics
(Fig. 1). All maps of the KKT region were created using
QGIS 3.10.1 with GRASS 7.8.1 (GRASS Development
Team, 2020; QGIS.org, 2020).
Collecting was done during the SokhoBio (Sea of
Okhotsk Biodiversity Studies, Malyutina et al., 2015)
campaign on board the RV Akademik M.A. Lavrentyev
in 2015 and the KuramBio II (Kuril Kamchatka
Biodiversity Studies II, Brandt, 2016) campaign on
board RV Sonne. All samples we refer to were collected
with a box corer (BC), an Agassiz trawl (AGT), and
two types of epibenthic sledges (Brandt et al., 2013;
Brenke, 2005; see Supplemental Table S1 for station
data). Collected sediment was sieved using 300 mm-
meshed metal sieves and filtered with 20 C precooled,
filtered seawater to remove fine sediment fractions. To
preserve the specimens for genetic studies all samples
were bulk-fixed in chilled 96% ethanol and kept chilled
at all times following Riehl et al. (2014). Using a
stereomicroscope Leica M60, all available haploniscid
specimens were sorted into taxonomic groups up to spe-
cies level by phenotypic clustering.
Voucher specimens for molecular analysis were selected
during the sorting procedure to represent the entire variabil-
ity of haploniscid phenotypic clusters (see below) and
cover their largest possible geographic distribution; they
were stored in individual cryovials containing 96% ethanol.
All voucher specimens have been photographed using
a Leica M125 C stereomicroscope equipped with a
Leica DMC 4500 camera and are part of a working col-
lection at the Senckenberg Museum in Frankfurt,
Germany (SGN). All material will be deposited at SGN
Fig. 1. Sampling area map (Kuril-Kamchatka Trench region). Overview of sampling areas of the KuramBio II (KBII) and SokhoBio
(SKB) expeditions in the greater Kuril-Kamchatka Trench region of the North-west Pacific, divided into four different geographic
zones following Johannsen et al. (2020).
Evolution and biogeography of Haploniscus (Isopoda) 3

and the Zoological Museum Hamburg, Germany in the
course of future taxonomic studies (see Supplemental
Table S2). DNA sequencing reads and contig sequences
were registered in the Barcode of Life Data System
(BoLD System, Ratnasingham & Hebert, 2007), avail-
able at dx.doi.org/10.5883/DS-NWPHA22, together with
voucher photographs and sampling metadata. From
there, sequence data were uploaded to GenBank
(Benson et al., 2013), accession numbers OM782497 to
OM782662. Locality data were uploaded to the Ocean
Biogeographic Information System (OBIS, Grassle,
2000), available at https://ipt.iobis.org/obis-deepsea/
resource?r=deep-sea_haploniscid_isopods. All align-
ments and primary phylogenetic trees were registered in
Zenodo (European Organization For Nuclear Research
& OpenAIRE, 2013) available at http://doi.org/10.5281/
zenodo.6553796.
Taxonomic feedback loop
To corroborate the stated primary hypothesis, morpho-
logical and molecular evidence was scrutinized with focus
on the belyaevi complex using a taxonomic feedback loop
(Page et al., 2005), a process also referred to as reciprocal
illumination (Hennig, 1965). As a first step, phenotypic
clusters among the belyaevi complex were identified by
morphological similarity using diagnostic characters
extracted from the literature and reconstructing the post-
marsupial development. In the second step, congruence
between genotypic and phenotypic clusters was searched
for using two standard mitochondrial markers (16S, COI).
Finally, operational criteria derived from the phylogenetic
species concept (PSC) introduced by Eldredge and
Cracraft (1980) and the unified species concept (USC)
introduced by De Queiroz (2007) were applied, synthesiz-
ing morphological, molecular, and biogeographic informa-
tion to determine species identities. The USC renders all
properties treated as secondary species criteria in other
species concepts relevant to species delimitation to the
extent that they provide evidence of lineage separation.
Morphological species delimitation and
identification
All available haploniscid isopods from the NWP were
analysed morphologically, with a focus on the six
phenotypic clusters of the Haploniscus belyaevi species
complex identified by Johannsen et al. (2020) delineat-
ing species also considering their phenotypic plasticity.
For identification of the species, original taxonomic lit-
erature (Birstein, 1963a,1963b,1971) and type material
housed at the Zoological Museum of Moscow
Lomonosov State University (MSU) were consulted.
This approach allowed the inclusion of further previ-
ously overlooked members of the species complex.
First, to resolve phenotypic plasticity and stage-
dependent sexual dimorphism, specimens were allocated
to ontogenetic stages, following the nomenclature and
characters used by Br€
okeland (2010b). Staging was
based on developing morphological character states, like
those of pereonite 7 and pereopods VII as well as male
pleopods I and II. In this study, the terms ‘ontogenetic
stage’and ‘instar’are distinguished from each other: An
ontogenetic stage includes all specimens that share a
certain combination of unique morphological character
states that undergo developmental changes when transi-
tioning into another such stage; an instar refers to a cer-
tain moult that may occur either within or between
ontogenetic stages –it is distinguishable from other
instars primarily by differences in body length. Body
length measurements were taken by sketch-drawing the
specimens laterally under a Leica M60 microscope with
camera lucida and using a micrometer for calibration.
Digitalized drawings were measured from the tip of the
rostrum to the tip of the posterolateral pleotelson proc-
esses under consideration of potential body curvature
using the measuring tool of Adobe Acrobat Reader. The
relative length of the posterolateral protrusions was cal-
culated as the posterolateral protrusion length (postero-
lateral protrusion tip to posterior pleotelson apex)
divided by the total pleotelson length (posterolateral pro-
trusion tip to posterior margin of pereonite 7).
Confocal Laser Scanning Microscopy (CLSM) was
used as a non-destructive alternative to scanning elec-
tron microscopy to study detailed morphological differ-
ences between phenotypic clusters and ontogenetic
stages. The ventral pleotelson was scanned for every
available ontogenetic stage of each phenotypic cluster
and dorsal habitus scans were additionally taken for the
adult male stage. Specimens were chosen on the premise
that molecular data were available, and least damaged
specimens were preferred. Single specimens of each
available ontogenetic stage (see Supplemental Table S3)
were transferred into glycerine and simultaneously
stained using Congo red dissolved in 70% denatured
ethanol following Michels and B€
untzow (2010). The
specimens were incubated for 3 days under dark condi-
tions to allow the ethanol to evaporate slowly to avoid
shrinking. Afterwards, the specimens were placed on
temporary slides following the technique established by
Michels and B€
untzow (2010) with slight modifications.
Given the large size of the specimens, sealing rings with
quadrate cross-section were extracted from cryovial lids
and used as spacer. For fixation of these rings and cov-
erslips (No. 1.5, 170 mm) clear nail polish was used.
Scans were conducted using a Leica DM2500 with a
4 H. Knauber et al.

Leica TCS SPE II and the LEICA LAS X 3.5.5.19976
software at a resolution of 2480 2480 pixels (see
Supplemental Table S4). Fiji ImageJ 1.52p (Schindelin
et al., 2012; Schneider et al., 2012) was used for initial
post-production, Adobe Photoshop CC 2019 20.0.3 and
Adobe Illustrator CC 2019 23.0.2 were used for final
adjustments. Panorama Stitcher Mini version 1.1.0
(Boltnev & Kacher, 2010) and the Image Composite
Editor (ICE) version 2.0.3.0 (Microsoft Corporation,
2015) were used for photo stitching.
DNA extraction, sequencing, and alignment
For DNA extraction up to four pereopods were dissected
from each specimen and were transferred from ethanol
to 20 ml PVP (#LGC Genomics) buffer and stored at
20 C. To prevent contamination with foreign DNA
preparation tools were heat-sterilized and preparation
dishes bleached and cleaned. In total, 149 specimens of
the hypothesized belyaevi complex were selected for
molecular analysis (Supplemental Table S2), alongside
139 additional samples of other haploniscid taxa from
the KKT region.
For molecular analyses (i) the (partial) mitochondrial
large ribosomal RNA subunit (16S) and (ii) the (partial)
cytochrome-c-oxidase subunit I (COI) were used follow-
ing principal directions provided by Riehl et al. (2014).
The PCR and subsequent Sanger DNA sequencing
(Sanger et al., 1977) were conducted at LGC Genomics
Germany, Berlin. The chosen primers for the 16S gene
were 16S SF and SR, while the dgLCO1490 and
dgHCO2198 primers were used for the COI gene frag-
ment (see Supplemental Table S5). Assembly, trimming
and multiple sequence alignments were performed in
Geneious 9.1.8 (https://www.geneious.com). The
Geneious plug-in MAFFT v7.308 (Katoh et al., 2002;
Katoh & Standley, 2013) was employed for multiple
sequence alignment with the settings L-INS-i, 200PAM/
k¼2, 1.53 gap open penalty and 0.123 offset. Multiple
sequence alignments were visually quality controlled
and, in the case of COI, checked for stop codons using
the amino-acid translation (invertebrate mitochondrial).
The aligned datasets were concatenated using the appro-
priate function in Geneious.
Sequences of the isopod families Desmosomatidae Sars,
1899, Macrostylidae Hansen, 1916 and Munnopsidae
Lilljeborg, 1864 from GenBank were used as outgroup
(see Supplemental Table S6).
Molecular species delimitation
To delineate genotypic clusters (i.e., genetically distinct
groups with few or no intermediates to other such
groups; Mallet, 1995) within the hypothesized belyaevi
complex, the distance-based “Assemble Species by
Automatic Partitioning”(ASAP; Puillandre et al., 2021)
and the tree-based bPTP (Zhang et al., 2013) were used
via their respective web tools. For the ASAP web tool,
which clusters molecular sequences into distinct groups
by applying different thresholds over multiple partitions,
16S and COI were analysed separately and the best-
suited models were selected based on their ASAP score
and number of delineated species in comparison to other
performed methods of species delimitation
(Supplemental Figs S1 and S2). Thresholds for molecu-
lar distance-based species delimitation from previous
studies on haploniscids (see Brix et al., 2011;Br
€
okeland
& Raupach, 2008), related isopods (S. Bober et al.,
2018; Brix et al., 2011,2015;Br
€
okeland & Raupach,
2008; Riehl & K€
uhn, 2020) as well as universal thresh-
olds (Hebert et al., 2003) were used as orientation, well
aware that any published threshold between intraspecific
variability and interspecific divergence is somewhat
arbitrary (hence the use of ASAP; Puillandre
et al., 2021).
We analysed the concatenated tree in the bPTP web
tool, which models branching events based on the num-
ber of mutations to distinguish sequence clusters, and
focused on the maximum likelihood results (mPTP).
For graphically differentiating between intraspecific
variability and interspecific divergence and the resulting
barcoding gap, histograms were produced with
Microsoft Excel, using a randomly chosen representative
for each phenotypic cluster (SKB Hap50, KBII Hap201,
and SKB Hap20) as comparison with all other sequen-
ces. For this purpose, matrices of pairwise distances
were calculated separately for 16S and COI alignment
in MEGA-X 10.1.18 (Kumar et al., 2018).
TCS haplo-networks confined to specimens of the
belyaevi complex and another closely related species
were calculated for each of the 16S and COI alignments
respectively using the software PopART 1.7 (Clement
et al., 2002; Leigh & Bryant, 2015). A geotag including
sampling area and region data was given to each ana-
lysed sequence to identify potential biogeographic pat-
terns. Phylogenetic analyses were performed in IQTree
2.0.6 for all three alignments (Minh et al., 2020), further
utilizing partition models in the case of the COI- and
the concatenated alignment (Chernomor et al., 2016).
For the latter, three independent partitions were ana-
lysed: the 16S gene fragment (partition 1), the COI first
and second codon positions (partition 2) and COI third
codon position (partition 3), to accommodate for differ-
ent mutation rates. The resulting phylogenetic trees were
illustrated using Interactive Tree of Life 5.6.2 (iTOL,
Letunic & Bork, 2007). The statistical support for each
Evolution and biogeography of Haploniscus (Isopoda) 5

node was evaluated based on the thresholds proposed in
Hillis and Bull (1993): bootstrap (bt) values exceeding
90 indicated high support (herein also called “strong”),
while bt values of 70–89 were considered well-sup-
ported. Weak support was assigned to 50–69, and bt
values below 50 did not indicate any support.
Results
Morphological species delimitation
The haploniscid samples from the two recent expedi-
tions in the NWP allowed for the discrimination of eight
phenotypic clusters belonging to the genus Haploniscus,
two phenotypic clusters of Hydroniscus and three
Mastigoniscus clusters (Supplemental Table S2). While
five of these are considered to represent undescribed
species, the remaining eight clusters could be allocated
to known haploniscid species from the NWP based on
the available original taxonomic literature (Birstein,
1963a,1963b,1971). These known species comprise
Haploniscus gibbernasutus Birstein, 1971,H. hydronis-
coides Birstein, 1963a,H. menziesi Birstein, 1963a,H.
profundicolus Birstein, 1971,H. ultraabyssalis Birstein,
1963b,Hydroniscus minutus Birstein, 1971,Hydr. vit-
jazi Birstein, 1963a, and Mastigoniscus latus
(Birstein, 1971).
Investigations of the MSU collection material
revealed severe mismatches between the original species
description and the presumed type material of
Haploniscus belyaevi. The latter comprised multiple
haploniscid species of which none could be allocated to
H. belyaevi as they did not match the original descrip-
tion (including illustration), and the exact syntype
depicted in the original species description was missing.
The available type material furthermore stems from a
sampling station which does not align with the station
data listed in the original description. As the where-
abouts of the actual type specimens are currently
unknown to the authors and could not be clarified upon
request by the responsible institution’s collection per-
sonnel it has to be assumed that the actual syntypes of
H. belyaevi have been lost. Accordingly, the identifica-
tion of H. belyaevi was based solely on the original
description text and illustrations (Birstein, 1963a).
With the aid of original literature regarding H.
belyaevi, initially three phenotypic clusters were differ-
entiated among the material from recent expeditions that
together form the belyaevi complex. All three pheno-
typic clusters shared two antennal spines, which were
located on the antennal third (basis) and fifth peduncular
articles (merus) respectively. It was found that the spine
on the third article is located dorsally and has a blunt,
triangular shape, while the spine on the fifth article is
located distodorsally and had an acute, elongate shape.
Further, all detected phenotypic clusters shared an acute
rostrum, yet the orientation of the rostrum varied
between clusters. These clusters differed especially with
regard to character states showing pronounced sexual
dimorphism in adult male specimens. The first pheno-
typic cluster was provisionally named Haploniscus
‘KKT’after its main distribution in the Kuril-
Kamchatka Trench. Morphological characters distin-
guishing H.‘KKT’from the other two phenotypic clus-
ters among the recently collected material and the
original H. belyaevi description included posterior exten-
sions of the pleotelson posterior margin in subadult and
adult males, dorsally “roofing”the uropods (Fig. 2.1),
and an anteriorly oriented rostrum with antero-dorsally
pointing tip. Additionally, the lateral pleotelson outlines
were straight while the pleotelson was narrowing poster-
iorly. Within this cluster, variation in the length of the
antennal distodorsal spine of the fifth article was found.
Further, the basis of the rostrum showed varying levels
of expression of a dorsal bulge. A re-examination of H.
‘KKT’following the detection of multiple distinct geno-
typic clades within this phenotypic cluster (see below)
revealed that hadal specimens lacked this dorsal bulge
at the basis of the rostrum, as opposed to abyssal speci-
mens. Also, the posterior pleotelson extensions were
more pronounced in hadal than in abyssal specimens
(data not shown). Accordingly, H.‘KKT’could be sub-
divided into the clusters H.‘KKT abyssal’and H.‘KKT
hadal’based on their depth distribution (see below).
The second phenotypic cluster, provisionally named
Haploniscus ‘SO’after its main region of occurrence in
the Sea of Okhotsk, was distinguishable from H.‘KKT’
solely by the shape of the pleotelson and its posterolat-
eral protrusions. While the lateral pleotelson outline was
straight anteriorly, it was convex posteriorly with longer
protrusions than in H.‘KKT’, also featuring a peculiar
laterally curved appearance. Overall, the shape of the
entire pleotelson was square rather than tapering in this
cluster, which was especially apparent in adult male
specimens (Fig. 2.2). Rostral morphology of H.‘SO’
did not vary from that of H.‘KKT’.
The third phenotypic cluster was identified as
Haploniscus aff. belyaevi as it matched the original spe-
cies description and illustration of H. belyaevi.
However, H. aff. belyaevi solely occurred in the Kuril
Basin of the SO and did not occur south of the KIR,
where H. belyaevi had been originally recorded (CTH,
ETA, WTA) as inferred from the original species
description (Birstein, 1963a). An unequivocal identifica-
tion was not possible because of the missing type mater-
ial. It differed from the before-mentioned phenotypic
6 H. Knauber et al.

clusters in the shape of the rostrum and the posterolat-
eral pleotelson protrusions. H. aff. belyaevi had an acute
rostrum with an anterior orientation. Over their ontogen-
etic development, the posterolateral protrusions of the
pleotelson reached a longer relative length (0–0.52 pleo-
telson length) than in the other two clusters (0–0.2 in H.
‘KKT’and 0–0.26 in H.‘SO’), which was particularly
noticeable in the adult males (Fig. 2.3). Thus, the pleo-
telson appeared somewhat square rather than tapering in
this cluster as well, which was, again, more pronounced
in adult male specimens.
Reconstruction of post-marsupial
development
Ontogenetic reconstruction of the three phenotypic clus-
ters resulted in the identification of three manca stages
(manca I–III, Fig. 3), three male stages (juvenile #IV,
preparatory #V, and copulatory #VI, Fig. 4), and two
female stages (preparatory $IV, ovigerous $V, Fig. 5).
A whole set of these ontogenetic stages was only pre-
sent for H.‘KKT’, while samples of H.‘SO’and H.
aff. belyaevi lacked two and three stages, respectively.
Whether these stages are non-existent or rather
unsampled remains unresolved. Several morphological
characters were identified that help distinguishing
between ontogenetic stages, regardless of the morpho-
logical variation between the three phenotypic clusters
(see Supplemental Fig. S1).
The manca I stage was recognizable by the absence
of the seventh pereonite and pereopods (Figs 3.1,3.4
and 3.7). During the manca II stage the anlagen of the
seventh pereopods developed beneath the cuticle of the
anteroventral pleotelson (Figs 3.2,3.5 and 3.8). In the
manca III stage the seventh pereopods have emerged
and are incompletely developed. They adopted a pos-
ition close to the medioventral body with merus through
dactylus paired up, apparently bearing no locomotive
function yet. Also, the setation of these limbs was
absent or not yet fully developed in this stage. The
seventh pereonite was incompletely developed in this
Fig. 2. CLSM images of adult males within the Haploniscus belyaevi species complex. Dorsal habitus CLSM scans of copulatory
males from three different phenotypic clusters within the H. belyaevi species complex: H.‘KKT’(KBII Hap165 (1)), H.‘SO’(SKB
Hap06 (2)), and H.aff.belyaevi (SKB Hap54 (3)).
Evolution and biogeography of Haploniscus (Isopoda) 7

stage as well, with the tergites having a cone-like shape
rather than a rectangular shape like all remaining ter-
gites (Figs 3.3,3.6 and 3.9). All subsequent stages of
the males (male IV–VI) were recognizable by the
presence of the first pleopods, which first appeared in
the juvenile #IV stage, projecting to half the length of
the still fused pleopods II (Figs 4.1 and 4.4). In the pre-
paratory #Vstage, pleopods I projected beyond the
Fig. 3. Ontogenetic development of the manca stages of the Haploniscus belyaevi species complex. Ventral pleotelson CLSM-scans
of three different phenotypic clusters H.‘KKT’(KBII Hap227 (1), KBII Hap234 (2), KBII Hap107 (3)), H.‘SO’(SKB Hap45 (4),
SKB Hap12 (5), SKB Hap39 (6)) and H.aff.belyaevi (SKB Hap09 (7), SKB Hap30 (8), SKB Hap52 (9)) across the three successive
ontogenetic stages: manca I (1, 4, 7), manca II (2, 5, 8) and manca III (3, 6, 9). Arrows on manca II specimens indicate the
subcuticular anlagen of the developing seventh pereopods.
8 H. Knauber et al.

now separated pleopods II. Further, the pleopod II endo-
pod (stylet) was visible, extending beyond the posterior
margin of the first pleopods (Fig. 4.2). In the copula-
tory #VI stage the first pleopods were characterized
by distolateral protrusions (lobes) (Figs 4.3,4.5 and
4.6). In adult females two morphologically different
stages were identified: The preparatory $IV stage
(Figs 5.1–5.2,5.4–5.6) was distinguishable from the
ovigerous $Vstage (Fig. 5.3) by the absence of a
marsupium. Instead of gradually developing oostegites
with external buds, as in Munnopsidae (Wilson, 1981),
the haploniscid isopods analysed here must either
develop their oostegites in a single moult or carry their
developing oostegite anlagen internally, as is the case in
Urstylidae (Riehl et al., 2014).
Overall, each detected phenotypic cluster is charac-
terized by at least some form of sexual dimorphism.
These differences between sexes were particularly
Fig. 4. Ontogenetic development of the adult male stages of the Haploniscus belyaevi species complex. Ventral pleotelson CLSM-
scans of three different phenotypic clusters H.‘KKT’(KBII Hap119 (1), KBII Hap216 (2), KBII Hap165 (3)), H.‘SO’(SKB Hap19
(4), SKB Hap06 (5)) and H.aff.belyaevi (SKB Hap54 (6)) across the three successive ontogenetic stages: juvenile male IV (1, 4),
preparatory male V (2) and copulatory male VI (3, 5, 6).
Evolution and biogeography of Haploniscus (Isopoda) 9

pronounced in the pleotelson posterior margin, includ-
ing enlarged posterolateral outgrowths in males of H.
‘SO’and H. aff. belyaevi (Figs 2.2 and 2.3) and mar-
ginal protrusions dorsally covering the male uropods of
H. ‘KKT’(Fig. 2.1). As opposed to the males, their con-
specific females are characterized by smaller outgrowths
or the absence of these protrusions respectively (Figs
5.2,5.5 and 5.6), resulting in a much higher interspe-
cific similarity.
Body length analysis of the ontogenetic
stages of the belyaevi complex
Analysing the body-length distribution of all available
intact specimens of the three phenotypic clusters H. aff.
Fig. 5. Ontogenetic development of the adult female stages of the Haploniscus belyaevi species complex. Ventral pleotelson CLSM-
scans of three different phenotypic clusters H.‘KKT’(KBII Hap123 (1), KBII Hap219 (2), KBII Hap118 (3)), H.‘SO’(SKB Hap41
(4), SKB Hap23 (5)) and H. aff. belyaevi (SKB Hap50 (6)) across the three successive ontogenetic stages: preparatory female IV
early instar (1, 4), preparatory female IV late instar (2, 5, 6) and ovigerous female V (3). The female IV stage is depicted twice to
compare specimens of its first and last instar. Depicted differences in shape and size between these instars are the result of a gradual
development over multiple moults that did not justify the erection of additional stages. The ovigerous female V specimen of H.
‘KKT’could not be scanned in a standardized view without the risk of damaging the specimen due to a pronounced body curvature.
10 H. Knauber et al.

belyaevi (N ¼14), H. ‘SO’(N ¼36) and H. ‘KKT’
(N ¼51) revealed similar size ranges for all groups
across their entire ontogenies, namely 1.2–4.0 mm,
1.1–4.4 mm and 1.2–4.5 mm respectively (Fig. 6). After
molecular analyses detected multiple genotypic clusters
within H. ‘SO’and H. ‘KKT’(see below) their body-
length histograms were subdivided accordingly. Within
H. ‘KKT’the ‘KKT hadal’group (N ¼29) reached
larger body sizes than the ‘KKT abyssal’one (N ¼22)
in each of the ontogenetic stages, except for the prepara-
tory $IV stage. No size differences became apparent
between the three genotypic clusters of H. ‘SO’(N(SO-
KIR)¼27; N(SO-SO)¼6; N(SO-WTA)¼3). Overlaps in
body-size ranges between consecutive ontogenetic stages
occurred primarily between the first two manca stages of
all three clusters (N(manca I)¼21; N(manca II)¼9) and
between the adult female stages of H. ‘KKT’(N($
IV)¼11; N($V)¼6). The largest size range within the
same ontogenetic stage was recorded for the preparatory
$IV stage in all phenotypic clusters (N¼25), which also
contained the largest specimens of H.‘SO’.Severalonto-
genetic stages exhibited at least one gap in their length
spectrum, if not multiple, which could indicate the exist-
ence of additional instars within each stage. This espe-
cially applied to the preparatory $IV stage (N ¼25), the
manca I stage (N ¼21) and, in the case of H. ‘KKT’,to
the male VI (N ¼9) and ovigerous $V stages (N ¼6).
The body-length histograms of multiple stages (manca
I, female IV, female V) suggest the existence of differ-
ent moults within these stages, that could not be differ-
entiated using our approach. An overlap of the size
ranges of manca I and manca II specimens, for instance,
highlights that either size variation is significant within
stages or that the onset of development for certain mor-
phological traits —in this case the subcuticular anlagen
of the seventh pereopods —is independent of body
size, or there may be a certain degree of flexibility as to
the moult in which these morphological traits occur.
Sequencing and alignment results
Altogether, tissue for the extraction of total genomic
DNA was dissected from 282 haploniscid specimens.
For 16S, 109 out of 166 sequences belong to one of the
three phenotypic clusters: H.‘KKT’(N ¼52), H.‘SO’
(N ¼42), and H. aff. belyaevi (N ¼14). Other known
haploniscid species present in this dataset were H.
hydroniscoides,H. menziesi,H. profundicolus and Hydr.
minutus. The genus Mastigoniscus was represented by
M. latus and M. sp. 1. Further information regarding
the 16S and COI alignments as well as the results of the
phylogenetic tree calculation are provided in the
Supplemental Table S7.
Out of 131 COI sequences, 72 belong to the species
complex of interest, including H.‘KKT’(N ¼52), H.
‘SO’(N ¼12), and H. aff. belyaevi (N ¼8). All haplonis-
cid species and phenotypic clusters from which 16S data
were successfully retrieved were also present in the COI
dataset, except H. menziesi and H. profundicolus.Nostop
codons were reported within the translated amino-acid
sequence. The amplification of the 16S marker (58.9%)
was more successful than the COI marker (46.5%). While
sequences of both investigated genes could be acquired
for most sampled isopods, some specimens were solely
represented by either a 16S or a COI sequence
(Supplemental Table S2). The concatenated 16S and COI
multiple-sequence alignment contained 186 sequences of
which 180 belong to haploniscid isopods and six to out-
group representatives.
Genetic variation and barcode-gap analysis
The histogram of pairwise inter- and intraspecific distan-
ces in the 16S alignments depicted a ‘barcoding gap’
separating the belyaevi complex’s phenotypic clusters
from each other (Fig. 7.1). Intraspecific variation within the
belyaevi complex was low (H. KKT: 1.3%; H. SO: 2.1%)
to absent (H. aff. belyaevi:0%;Table 1), while interspe-
cific divergence between the clusters of the belyaevi com-
plex ranged between 5.9% and 8.9%, with most other
haploniscid species from the KKT region being divergent
by more than 17.8% from the belyaevi complex’sclusters
(Table 2). Haploniscus profundicolus represents the sole
exception to this finding, given its close relationship with
H. KKT (2% divergence, Fig. 7.1), despite not being con-
sidered as a potential member of the belyaevi complex ini-
tially due to morphological differences.
Within the COI dataset, levels of intraspecific variation
and interspecific divergence were generally higher and, in
many cases, up to twice as high than in the 16S dataset
(Tables 1 and 2). Consequently, intraspecific variation
within H.‘KKT’and H. ‘SO’reached maximum p-dis-
tances of 10.8%, while variation within H. aff. belyaevi
remained comparatively low with maximum levels of up
to 1.7% (Table 1). The interspecific divergence between
the clusters of the belyaevi complexrangedbetween
13.2–21.6% in COI (Fig. 7.2), while all other haploniscid
species were divergent by more than 22% (Table 2).
In both markers the intraspecific variation of H.
‘KKT’was distributed across several discrete peaks,
indicating the existence of at least two distinct geno-
typic clusters at 0.5%, 1.5% and 9.5% divergence (Fig.
7.2). Similar findings were recorded within H.‘SO’,
whose distribution of variability showed three peaks:
between 0.5% and 1.5% (overlapping distributions) and
5.5 as well as 10.5% (discrete, Fig. 7.2). In both
Evolution and biogeography of Haploniscus (Isopoda) 11

Fig. 6. Ontogenetic body-length frequency of the Haploniscus belyaevi species complex. Bodylength-frequency histograms of
ontogenetic stages for three analyzed phenotypic clusters (H. aff. belyaevi,H.‘KKT’and H.‘SO’) and genotypic clusters within.
Stages represented include manca I–III, juvenile #IV, preparatory #V, copulatory #VI, preparatory $IV and ovigerous $V.
12 H. Knauber et al.

Fig. 7. Barcoding–gap analysis of the Haploniscus belyaevi species complex. Stacked (i.e., placed on top of each other) histograms
contrasting the intraspecific variations of the phenotypic clusters H.aff.belyaevi (SKB Hap50), H.‘KKT’(KBII Hap201), H.‘SO’
(SKB Hap20) with their interspecific divergences (also considering H.profundicolus) based on uncorrected p-distances of the 16S
alignment (1) and the COI alignment (2).
Table 1. 16S and COI intracluster p-distances. Intraspecific variation (mean uncorrected p-distances and
range in brackets) within each genotypic cluster for the 16S and the COI gene respectively. N/A denotes
cases in which calculation of p-distance was not possible due to low sequence counts.
Cluster p-distance 16S p-distance COI
Haploniscus aff. belyaevi 0.000 (0.000) 0.006 (0.000–0.017)
Haploniscus 'KKT' 0.005 (0.000–0.013) 0.051 (0.000–0.108)
KKT abyssal 0.000 (0.000) 0.011 (0.000–0.027)
KKT hadal 0.000 (0.000–0.003) 0.007 (0.000–0.018)
HT 13 N/A N/A
HT 14 0.000 (N/A) 0.006 (N/A)
Haploniscus 'SO' 0.005 (0.000–0.021) 0.058 (0.000–0.108)
SO-KIR 0.001 (0.000–0.008) 0.008 (0.002–0.020)
SO-SO 0.001 (0.000–0.003) 0.004 (0.000–0.005)
SO-WTA 0.000 (0.000) 0.003 (0.000–0.005)
Haploniscus profundicolus N/A N/A
Haploniscus menziesi 0.107 (0.000–0.160) N/A
Haploniscus hydroniscoides 0.015 (0.000–0.056) 0.036 (0.000–0.121)
Hydroniscus minutus N/A N/A
Mastigoniscus latus 0.023 (0.000–0.104) 0.027 (0.000–0.130)
Mastigoniscus sp. 1 0.003 (0.000–0.008) 0.021 (0.000–0.040)
Evolution and biogeography of Haploniscus (Isopoda) 13

Table 2. 16S and COI intercluster p-distances. Mean uncorrected p-distances between each genotypic cluster of the H. belyaevi species complex and other species of the
genus Haploniscus for 16S (below diagonal) and COI data (above diagonal) respectively, representing mean interspecific divergence. N/A denotes cases in which
calculation of p-distance was not possible due to the unavailability of molecular sequences for certain clusters and the respective gene.
COI
16S H. aff.
belyaevi
H. 'KKT' H. 'SO'
H.
profundicolus
H.
menziesi
H.
hydroniscoides
KKT
abyssal
KKT
hadal HT 14 HT 13 SO-KIR SO-SO SO-WTA
H. aff. belyaevi 0.174
(0.169–
0.184)
0.181
(0.174–
0.199)
0.177
(0.170–
0.186)
0.181
(0.177–
0.190)
0.144
(0.132–
0.158)
0.149
(0.139–
0.165)
0.153
(0.139–
0.178)
N/A N/A 0.238
(0.224–
0.274)
H. 'KKT'
KKT abyssal 0.083
(0.083–
0.084)
0.083
(0.073–
0.097)
0.099
(0.094–
0.108)
0.098
(0.095–
0.102)
0.197
(0.187–
0.204)
0.183
(0.174–
0.193)
0.184
(0.180–
0.188)
N/A N/A 0.250
(0.239–
0.273)
KKT hadal 0.080
(0.080–
0.083)
0.008
(0.008–
0.008)
0.089
(0.081–
0.100)
0.078
(0.074–
0.084)
0.203
(0.196–
0.216)
0.187
(0.177–
0.197)
0.193
(0.187–
0.200)
N/A N/A 0.247
(0.240–
0.267)
HT 14 0.085
(0.085–
0.086)
0.013
(0.013–
0.013)
0.010
(0.010–
0.013)
0.082
(0.079–
0.086)
0.181
(0.175–
0.188)
0.166
(0.161–
0.170)
0.172
(0.170–
0.174)
N/A N/A 0.250
(0.244–
0.274)
HT 13 0.088
(0.088–
0.089)
0.010
(0.010–
0.010)
0.008
(0.008–
0.010)
0.013
(0.013–
0.013)
0.196
(0.190–
0.204)
0.180
(0.179–
0.182)
0.195
(0.195–
0.195)
N/A N/A 0.255
(0.250–
0.265)
H. 'SO'
SO-KIR 0.068
(0.067–
0.073)
0.080
(0.080–
0.085)
0.080
(0.077–
0.085)
0.086
(0.082–
0.088)
0.083
(0.082–
0.088)
0.056
(0.048–
0.063)
0.101
(0.095–
0.108)
N/A N/A 0.245
(0.225–
0.271)
SO-SO 0.060
(0.059–
0.063)
0.078
(0.077–
0.080)
0.078
(0.075–
0.080)
0.083
(0.082–
0.085)
0.080
(0.080–
0.082)
0.009
(0.008–
0.015)
0.097
(0.091–
0.103)
N/A N/A 0.243
(0.235–
0.267)
SO-WTA 0.065
(0.065–
0.065)
0.077
(0.077–
0.078)
0.077
(0.075–
0.077)
0.082
(0.082–
0.082)
0.080
(0.080–
0.080)
0.016
(0.015–
0.021)
0.013
(0.013–
0.015)
N/A N/A 0.247
(0.240–
0.271)
H. profundicolus 0.087
(0.087–
0.087)
0.022
(0.022–
0.022)
0.019
(0.019–
0.019)
0.022
(0.022–
0.022)
0.028
(0.028–
0.028)
0.078
(0.085–
0.090)
0.075
(0.075–
0.078)
0.081
(0.081–
0.081)
N/A N/A
H. menziesi 0.195
(0.194–
0.198)
0.196
(0.193–
0.201)
0.196
(0.191–
0.206)
0.196
(0.191–
0.206)
0.190
(0.183–
0.204)
0.185
(0.178–
0.198)
0.186
(0.183–
0.188)
0.187
(0.186–
0.191)
0.217
(0.214–
0.224)
N/A
H. hydroniscoides 0.259
(0.253–
0.274)
0.245
(0.240–
0.268)
0.245
(0.237–
0.267)
0.244
(0.235–
0.270)
0.245
(0.240–
0.267)
0.263
(0.253–
0.289)
0.258
(0.252–
0.283)
0.258
(0.252–
0.279)
0.261
(0.258–
0.269)
0.255
(0.227–
0.286)
14 H. Knauber et al.

markers these discontinuities of the variability distribu-
tion were due to the same individuals assigned to H.
‘KKT’and H. ‘SO’.
Combined phylogenetic analysis
The comparison of the two resulting cladograms from
the 16S and COI alignments revealed congruent place-
ments for most identified clades within the phylogenetic
trees (see Supplemental Fig. S2), thus justifying a com-
bined analysis of a concatenated dataset. In both clado-
grams the family Haploniscidae as well as the three
phenotypic clusters of the belyaevi complex were mono-
phyletic. The belyaevi complex was paraphyletic in the
16S-based cladogram as H. profundicolus adopted a sis-
ter-group position to a clade comprising H. ‘KKT’and
H. aff. belyaevi while, unfortunately, no COI data for
this taxon were available. Incongruent placements of a
few branches can be explained by poor or lacking reso-
lution and resulting polytomies (Supplemental Fig. S2).
The consensus tree of the concatenated 16S-COI mul-
tiple-sequence alignments included 180 haploniscid speci-
mens which we divided into nine different monophyletic
groups representing (morphologically determined) known
haploniscid species from the KKT region as well as the
phenotypic clusters differentiated here, forming the
ingroup (Fig. 8).
Focusing on the latter, 112 sequences represented the
belyaevi complex as well as H. profundicolus, which
was placed among the belyaevi complex. Of the
belyaevi complex members, H.‘KKT’represented the
largest clade with 55 specimens. It formed a highly sup-
ported sister group (95 bt) to H. profundicolus.
Amongst H.‘KKT’, four highly supported clades can be
differentiated, of which a doubleton clade composed of
KBII Hap118 and KBII Hap207 (later called HT 14)
formed the most basal group. The relationships among
the rest of them remained unresolved (bt <50). The two
main groups of H.‘KKT’were preliminarily named H.
‘KKT abyssal’(96 bt) and H. ‘KKT hadal’(93 bt),
according to their (main) depth distribution.
The second clade among the belyaevi complex was
reasonably well supported (75 bt) and included H. aff.
belyaevi (14 specimens; 100 bt) and H.‘SO’(42 speci-
mens; 90 bt) (Fig. 8). Among H. aff. belyaevi no dis-
tinct clades became apparent due to highly similar
terminals. Among H.‘SO’, however, three highly sup-
ported clades stood out that were provisionally named
according to their (main) areas of occurrence (H. ‘SO-
SO’,H. ‘SO-KIR’, and H. ‘SO-WTA’). While H. ‘SO-
WTA’(98 bt) has the basal position, H. ‘SO-SO’(95
bt) and H. ‘SO-KIR’(100 bt) form a highly supported
(93 bt) sister-group relationship.
Overall, the phylogenetic analysis of the concatenated
dataset resulted in similar topologies but better statistical
support for the basal nodes, thus solidifying the distinc-
tion of phenotypic clusters.
ASAP and mPTP analyses
The best partition of the ASAP-analysis for the 16S
gene (ASAP-16S) as inferred from its ASAP-score
(1.00) divided the belyaevi complex into three genotypic
clusters, that are in accordance with the phenotypic clus-
ters that form the belyaevi complex (Fig. 8). The sole
exception is the lumping of the single H. profundicolus
specimen with H. KKT into a single cluster. For the
best partition of the COI gene (ASAP-COI; ASAP-
score: 2.00) eight groups were delineated. The extra
subdivision of genotypic clusters was restricted to H.
SO and H. KKT, which comprise three and four groups
respectively. Regarding the tree-based mPTP approach
that was applied to the concatenated phylogenetic den-
drogram, nine distinct clusters were identified. As in
ASAP-COI, H. SO is further split into three clusters,
whilst H. aff. belyaevi remains a single cluster. A sub-
division of H. KKT into the same four groups as in
ASAP-COI is also present in the mPTP analysis. H.
profundicolus is separated from but sister to H. KKT.
Geographic distribution and molecular
variability of the belyaevi complex
In total, the 16S haplotype network contained 109
sequences which were divided into 15 haplotypes (HT;
Fig. 9.1). Aside from H. aff. belyaevi (14 sequences,
HT 1), H.‘SO’(42 sequences, HT 2–9), and H. ‘KKT’
(52 sequences, HT 11–15), the haplotype network also
contained the single sequence of H. profundicolus (1
sequence, HT 10) due to its close evolutionary relation-
ship to H. ‘KKT’(see below). The single specimen of
H. profundicolus (KBII Hap117) was not only closely
related to H. ‘KKT’(9 mutations), but also occurred in
sympatry with H. ‘KKT’at the KBII A8 sampling area
located within the ETA region. All remaining pheno-
typic clusters of the belyaevi complex were separated in
the haplotype analysis by high numbers of bp changes
in 16S. Among the five observed H. ‘KKT’haplotypes,
two haplotypes were dominating that have distinct geo-
graphic distributions: (i) HT 12 was primarily recorded
within the CTH region at hadal depths (5,493–8,191 m;
‘KKT hadal’) while (ii) HT 15 occurred at abyssal
depths (4,469–5,755 m) of the ETA and WTA regions
(hence called ‘KKT abyssal’). Co-occurrence between
both haplotypes was restricted to a single sampling area
(KBII-A10) in the WTA region and they differed by
Evolution and biogeography of Haploniscus (Isopoda) 15

five bp changes in 16S. HT 11 differed from HT 12 by
a single bp change in 16S and also occurred in the
CTH. The remaining HT 13 and HT 14 differed from
HT 12 by three bp changes in 16S each and occurred in
the CTH and ETA, respectively. H. aff. belyaevi was
collected from three different sampling areas in the SO
region (SKB-A2, A4, A11) and was represented by a
single 16S haplotype. All H. aff. belyaevi records cover
a narrow depth range of 3,210–3,366 m and exclusively
stem from the Kuril Basin. H. aff. belyaevi and H.‘SO’,
Fig. 8. Consensus phylogenetic tree (16S and COI) of 186 haploniscid isopods. Sequence allocation to distinct clusters based on
ASAP and mPTP analyses (white rings) and morphological identity (coloured background) is highlighted. Sequences are genetically
clustered based on the results of an ASAP-analysis for the 16S (circle) and COI gene (triangle) as well as a mPTP-analysis
(diamond). Shaded ring segments indicate gaps within a cluster due to missing molecular data of the specimens in question for
this gene.
16 H. Knauber et al.

which occurred sympatrically at several stations within
the Kuril Basin, were separated by a minimum of 23 bp
changes. The H.‘SO’phenotypic cluster showed the
highest 16S diversity and was grouped into eight 16S
haplotypes. This diversity was reflected in the biogeog-
raphy of the group, which occurred at altogether six
sampling areas covering a range from the Kuril Basin in
the SO region, across the KIR to the WTA region (Fig.
9.1). However, no geographic pattern became apparent
in the haplotype network. As in H. ‘KKT’, few main
haplotypes and multiple unique derivative haplotypes,
which differ from the main haplotypes by 1–2 muta-
tions, characterized the H.‘SO’network. HT 3 and HT
4 occurred within the WTA region at depths of 4,769
but were genetically distinct.
Given the degree of separation between the pheno-
typic clusters of the belyaevi complex in 16S, three sep-
arate haplotype networks were calculated based on 72
COI sequences. In comparison to the 16S haplotype net-
work these were characterized by a larger number of
Fig. 9. 16S (1) and COI (2) haplotype networks of the Haploniscus belyaevi species complex and H.profundicolus. Haplotypes are
colour-coded after their sampling area and geographic area following Johannsen et al. (2020). Circled numbers represent mutation
steps for improved clarity. HT, haplotype.
Evolution and biogeography of Haploniscus (Isopoda) 17

haplotypes (N ¼28) as well as a higher frequency of bp
changes within the investigated phenotypic clusters (Fig.
9.2). 16S haplotypes that were split into multiple COI
haplotypes are named accordingly to facilitate traceabil-
ity. The 52 COI sequences belonging to the H.‘KKT’
cluster (HT 12–17) showed the same geographic distri-
bution as their associated 16S-sequences. A geographic
structure that was already indicated in the 16S data
became even more clear here: a hadal group, with one
exception collected at the CTH region (‘KKT hadal’;
HT 12.1–12.6, HT 16) at 5,493–8,191 m depth, com-
prised two main haplotypes (HT 12.1, HT 12.2) and
several unique haplotypes which differed from either of
the main haplotypes by 1–4 bp changes; despite originat-
ing from the WTA sampling area KBII-A10, HT 12.7
was most closely related to the hadal HT 12.6 and HT
12.5. A second group, that differed from the ‘KKT
hadal’group by 30 or more bp changes occurred solely
at abyssal depths (4,469–5,755 m) of the ETA and WTA
regions (‘KKT abyssal’; HT 15.1–15.9, HT 17). Among
the ‘KKT abyssal’group no haplotypes were shared
between ETA and WTA and within both regions all
haplotypes were more similar to each other than
between the regions. The hadal HT 13 and the abyssal
HT 14 were unique and relatively distinct from any
other haplotype of the H.‘KKT’cluster. Haploniscus
aff. belyaevi (HT 1.1–1.4) was represented in the COI
haplotype network by eight sequences that were found
at two sampling areas within the SO region (SKB-A2
and SKB-A11). It featured one common haplotype that
was distributed in both sampling areas, as well as three
unique haplotypes from SKB-A11. The H. ‘SO’cluster
(HT 2, HT 3, HT 5.1–5.4, HT 6) comprised 12 sequen-
ces distributed across seven haplotypes. A clear geo-
graphic structure became apparent in this COI network
separating the haplotypes HT 2 from the SO region
(H.‘SO-SO’) and HT 3 from the WTA region (hence
H.‘SO-WTA’) from the remaining haplotypes of the H.
‘SO’cluster. These remaining haplotypes (HT 5.1–5.4,
HT 6) formed a group covering a large geographic
range from the SO across the KIR into the WTA (hence
H.‘SO-KIR’).
Discussion
Taxonomic feedback loop and synthesis
As the central unit in biology is the species, accurate
species delimitation is a critical first step upon which
subsequent organism-based research and conservation
depend. A pluralistic, integrative approach to tax-
onomy is essential for improved species delimitation
and discovery (Padial et al., 2010), especially in the
deep sea, where the vast majority of eukaryotic spe-
cies currently remain undiscovered (Cordier
et al., 2022).
Evidence presented here from multiple data sources
validates the hypothesis that Haploniscus belyaevi
sensu Johannsen et al. (2020) is a complex of closely
related species. The comparison of molecular and mor-
phological approaches revealed principal congruence
(Figs 7.1 and 9.1, Table 2) supporting the distinction
of three main lineages within this belyaevi complex.
Analyses of the partial COI gene, which as expected
exhibited a higher variability than 16S (see, e.g., Brix
et al., 2011; Riehl et al., 2014)(seeSupplemental Fig.
S2), revealed further subdivisions among these three
groups which in turn also allowed for the identification
of minor but consistent morphological differences
between some of the groups (compare H. KKT above).
While H. aff. belyaevi formed a distinct and coherent
group across all datasets and its full distribution range
(e.g., Fig. 9.2), the results were more complex for H.
‘SO’and H. ‘KKT’.
Molecular data support the separation of an abyssal
group from a mostly hadal group among H. ‘KKT’and
indicate the existence of an additional two rare species
that were clearly differentiated (Fig. 9.2). Re-scrutiniz-
ing the specimens morphologically, the initially delim-
ited phenotypic cluster H. ‘KKT’exhibited variation of
the rostral and pleotelson morphology as well as body
size corroborating the separation of an abyssal from a
hadal group (Fig. 6). In conjunction, the congruence
between genotypic and phenotypic clusters as well as a
highly supported phylogenetic distinction and minimally
overlapping geographic distributions provide sufficient
support for the delimitation of two separate species H.
‘KKT abyssal’and H. ‘KKT hadal’according to the
USC and PSC (De Queiroz, 2007; Eldredge & Cracraft,
1980). A morphological delineation was impossible for
the distinct haplotypes HT 13 and HT 14 (Figs 9 and
10) due to the small number of specimens available and
their respective ontogenetic stages (HT 13: manca; HT
14: adult $stages).
Within H. ‘SO’, molecular approaches support three
groups (Figs 7–9;Tables 1 and 2) with partially over-
lapping geographic occurrences. Only H. ‘SO-WTA’
and H. ‘SO-KIR’did not have any sympatric occurrence
in the samples (Fig. 9.1). Morphological re-examination
did not reveal noticeable differences between these
groups. According to the criteria postulated by Held
(2003) and summarized by Raupach and W€
agele (2006)
(i.e. (i) bimodal distribution of pairwise distance values
without intermediates, (ii) differentiation at a level
known for this gene from undisputed species pairs
closely related to the studied species and (iii) persistence
18 H. Knauber et al.

of high levels of genetic differentiation in sympatry) H.
‘SO-SO’,H. ‘SO-KIR’, and H. ‘SO-WTA’qualify as
cryptic species.
Applying morphological, genetic, phylogenetic, and
biogeographic criteria, six species can be recognized
among the belyaevi complex fulfilling the USC and the
PSC: (i) H. aff. belyaevi, (ii) H. ‘SO-KIR’, (iii) H. ‘SO-
SO’, (iv) H. ‘SO-WTA’, (v) H. ‘KKT abyssal’, and (vi)
H. ‘KKT hadal’. As for H. aff. belyaevi, we refrained to
recognize this species as H. belyaevi at this stage due to
Fig. 10. Phylogeography of the Haploniscus belyaevi species complex in the Kuril-Kamchatka Trench region. Morphological
diversity (bottom), simplified phylogenetic diversity (middle) and schematic geographic distribution (top) of each of the six distinct
species of the H.belyaevi complex are visualized.
Evolution and biogeography of Haploniscus (Isopoda) 19

the missing type material and the distribution differences
of the two species. Highly differentiated singleton and
rare haplotypes (H. profundicolus, HT 13 and HT 14)
indicate the existence of additional species within the H.
belyaevi complex. The variation of differentiation of
genotypic and phenotypic traits among its members
made it clear that no single dataset alone would have
been sufficient to discover the full species diversity of
the belyaevi complex. This highlights the strengths of an
integrative approach to taxonomy.
Species boundaries among the H.
belyaevi complex
Allometric and other morphological changes during
development, such as sexual dimorphisms, are common
among the animal kingdom, also in deep-sea Asellota
(e.g., S. Bober et al., 2018; Jennings et al., 2020), such
as Haploniscidae (Br€
okeland, 2010b,2010a). While
such morphological disparity between sexes and life
stages may cause taxonomic uncertainty (e.g., Riehl &
K€
uhn, 2020), in resolving the developmental patterns of
individual species lies great potential to infer and better
understand ecology and evolution of deep-sea organisms
that are otherwise difficult to study (see, e.g., S. Bober
et al., 2018; Kniesz et al., 2018).
It was a noteworthy observation by Johannsen et al.
(2020) that some of the phenotypic clusters comprised
either only a certain developmental stage or only one
sex (Johannsen et al., 2020), suggesting that phenotypic
plasticity, such as sexual dimorphism or ontogenetic
changes, hindered the allocation of conspecific males
and females. Our data show that the actual species
boundaries do not reflect those originally proposed by
Johannsen et al. (2020). The complex is composed of
even more (up to nine) species, including several mor-
phologically cryptic species and, surprisingly, potentially
H. profundicolus.
Ontogenetic reconstruction revealed three main
phenotypic groups within this species complex and
showed that during their development all of these spe-
cies undergo strong and sex-specific morphological
changes (Figs 2–5,Supplemental Fig. S1), causing diffi-
culties for morphology-based species identification. We
investigated their ontogeny with focus on general mor-
phological features known to change during post-marsu-
pial development, such as the seventh pereopods, the
seventh pereonites, or the male first pleopods
(Br€
okeland, 2010b; Hessler, 1970; Wolff, 1962). This
allowed us to improve our understanding of how sexu-
ally dimorphic characters develop throughout maturation
and, ultimately, to link conspecific instars (Figs 2–5,
Supplemental Fig. S1). Three species complexes had
been previously reported for the Haploniscidae
(Br€
okeland, 2010a;Br
€
okeland & Raupach, 2008; Paulus
et al., 2022). Similar to our findings, two of these are
also characterized by a somewhat pronounced and
unique sexual dimorphism, a prominent characteristic
also found in other haploniscids (e.g., Birstein, 1963a;
Br€
okeland, 2010b,2010a). In these cases, females share
a relatively common morphology across species while
morphological differentiation is pronounced in adult
males (Br€
okeland, 2010a; Knauber, unpublished data).
Tracing the morphological changes through post-marsu-
pial development was the only way to delineate species
groups on morphological grounds with certainty.
Because our findings for the belyaevi complex are
showing the same pattern we assume we are dealing
with a rather common phenomenon in the
Haploniscidae.
This family is not the only group of crustaceans in
the deep-sea benthos with strong morphological changes
during ontogeny affecting diagnostic characters (see,
e.g., Riehl & K€
uhn, 2020) or sexual dimorphism (e.g.,
J. Bober et al., 2019; Duncan, 1983; Riehl et al., 2012;
Rivera & Oakley, 2009). Given that any organism-cen-
tred, broad-picture seeking branch of comparative biol-
ogy relies on a meaningful taxonomic foundation and
the names it creates (Patterson et al., 2010; Sigwart
et al., 2021), our study adds to the growing body of lit-
erature highlighting the necessity for high-quality taxo-
nomic information that includes verified DNA reference
libraries as well as ontogenetic information (Korshunova
et al., 2017; Methou et al., 2020) in an integrative
approach to taxonomy.
The 16S data of H. profundicolus –initially included
for means of comparison –suggests that this species is
closely related to the belyaevi complex. Indeed, its sis-
ter-group relationship with H. ‘KKT’(Fig. 8) and high
molecular similarity with H. ‘KKT hadal’(0.02 uncorr.
p-distance; Fig. 7.1) suggests it is part of the same
phylogenetic lineage. However, H. profundicolus is
strikingly different morphologically: (i) the adult male
specimen lacks the posterior protrusions of the pleotel-
son covering the uropods and (ii) further lacks the disto-
dorsal spine on the fifth peduncular article of the second
antenna, which represents the character that distin-
guishes members of the belyaevi complex from other
haploniscids from the KKT region.
The close genetic affiliation of H. profundicolus and
the belyaevi complex was surprising. Over all, our
results suggest that the high morphological similarities
between the members of the belyaevi complex, in par-
ticular the juvenile stages and females, is due to a lack
of differentiation rather than chance similarities because
these species are phylogenetically relatively young (Fig.
20 H. Knauber et al.