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An integrative approach for species delimitation in the spider genus Grammostola (Theraphosidae, Mygalomorphae)

Authors:
  • 1- Department of Pest-management and Conservation / Faculty of Agriculture and Life Sciences Lincoln University // 2- Instituto de Investigaciones Biológicas Clemente Estable // 3- Facultad de Ciencias, UdelaR

Abstract and Figures

The mygalomorph genus Grammostola (family Theraphosidae) is endemic to South America. The species Grammostola anthracina is one of the largest spiders in Uruguay and reputed to be the longest lived tarantula in the world. This nominal species has two distinct colour morphs comprising black and reddish-brown forms with controversial taxonomic status. Here, we present a phylogenetic study based on molecular characters (cytochrome c oxidase subunit I) of haplotypes of G. anthracina and closely related species. Our analysis together with new morphological data and biogeographical information indicates that the two morphs of G. anthracina constitute different species that are not sister to each other. Consequently, a new species, Grammostola quirogai is described, diagnosed and illustrated to encompass the black morph. Phylogenetic relationships and new taxonomic characters for Grammostola species included in this study are discussed.
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An integrative approach for species delimitation in the spider
genus
Grammostola
(Theraphosidae, Mygalomorphae)
LAURA MONTES DE OCA,GUILLERMO D0EL
IA &FERNANDO P
EREZ-MILES
Submitted: 23 June 2015
Accepted: 21 October 2015
doi:10.1111/zsc.12152
Montes de Oca, L., D0El
ıa, G. & P
erez-Miles, F. (2015). An integrative approach for
species delimitation in the spider genus Grammostola (Theraphosidae, Mygalomorphae).
Zoologica Scripta,00, 000000.
The mygalomorph genus Grammostola (family Theraphosidae) is endemic to South America.
The species Grammostola anthracina is one of the largest spiders in Uruguay and reputed to
be the longest lived tarantula in the world. This nominal species has two distinct colour
morphs comprising black and reddish-brown forms with controversial taxonomic status.
Here, we present a phylogenetic study based on molecular characters (cytochrome coxidase
subunit I) of haplotypes of G. anthracina and closely related species. Our analysis together
with new morphological data and biogeographical information indicates that the two
morphs of G. anthracina constitute different species that are not sister to each other. Conse-
quently, a new species, Grammostola quirogai is described, diagnosed and illustrated to
encompass the black morph. Phylogenetic relationships and new taxonomic characters for
Grammostola species included in this study are discussed.
Corresponding author: Laura Montes de Oca, Laboratorio de Etolog
ıa, Ecolog
ıa y Evoluci
on,
Instituto de Investigaciones Biol
ogicas Clemente Estable, Av. Italia 3318, CP 11600, Montevideo,
Uruguay. E-mail: lmontesdeoca@iibce.edu.uy
Laura Montes de Oca, Laboratorio de Etolog
ıa, Ecolog
ıa y Evolucion, Instituto de Investigaciones
Biol
ogicas Clemente Estable, Av. Italia 3318, CP 11600, Montevideo, Uruguay. E-mail: lmontes-
deoca@iibce.edu.uy
Guillermo D0El
ıa, Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile,
Campus Isla Teja s/n casilla 567, Valdivia, Chile. E-mail: guille.delia@gmail.com
Fernando P
erez-Miles, Secci
on Entomolog
ıa, Facultad de Ciencias, Universidad de la Rep
ublica,
Igua 4225, CP 11600, Montevideo, Uruguay. E-mail: myga@fcien.edu.uy
Introduction
Although many species concepts (e.g. Coyne & Orr 2004)
exist, a general consensus that considers species as differ-
ently evolving metapopulation lineages (de Queiroz 1998,
2005, 2007) has developed. Typically referred to as the
Lineage Species concept, it treats other species concepts as
operational criteria used to identify and delimit species
level lineages (Wiens & Penkrot 2002; Sites & Marshal
2003; de Queiroz 2005, 2007). An integrative approach to
taxonomy, based on multiple lines of evidence, is thought
to be necessary due to the complexity inherent to species
delimitation (Dayrat 2005; Agnarsson & Kuntner 2007;
Padial et al. 2010; Schlick-Steiner et al. 2010), which is at
least in part due to the variable as well as non-stationary
nature of the species (see Hey et al. 2003).
In spiders, as in other groups, morphological characters
have been historically used to delimit species boundaries
(Bertani 2001), but in several cases relying exclusively on
morphology has proven to be insufcient. As such, molecular
data are sometimes used in spider taxonomy (Starret &
Hedin 2007; Bond & Stockman 2008; Aguilera et al. 2009;
Hendrixon et al. 2013; Hamilton et al. 2014) to delimit spe-
cies in particularly difcult groups like mygalomorph spi-
ders. Morphologic characters used in taxonomy of the family
Theraphosidae mostly come from the genitalia (e.g. P
erez-
Miles et al. 1996; Bertani 2000); however, in the theraphosid
genus Grammostola Simon, 1892; genitalia are extremely
homogenous across species, making species delimitation
based on these characters a difcult, if not impossible, task
(Bucherl 1957; Schiapelli & Gerschman 1979).
Grammostola is endemic to South America and comprises
20 nominal species (World Spider Catalog 2015). Taxo-
nomic studies of this genus are scarce and most were pub-
lished decades ago (e.g. Bucherl 1951; Schiapelli &
ª2015 Royal Swedish Academy of Sciences 1
Zoologica Scripta
Gerschman 1979; but see Ferretti et al. 2011, 2013).
Although seven species of the genus have been cited for
Uruguay (World Spider Catalog 2015), P
erez-Miles (1985)
has disregarded the presence in Uruguay of G. grossa (Aus-
serer 1871) and G. actaeon (Pocock 1903). In addition,
G. alticeps is only known from its type material referred as
from Loc. Uruguay; without further history(Pocock
1903). Therefore, only four species are well documented in
Uruguay: G. andreleetzi Vol 2008, Grammostola anthracina
(Koch 1842), Grammostola burzaquensis Ibarra 1946 and
Grammostola iheringi (Keyserling 1891) (Montes de Oca &
P
erez-Miles 2009). Grammostola anthracina is the only one
that has a wide distribution in the country. Unfortunately,
this species is threatened by habitat fragmentation and ille-
gal trafc to European countries associated with the pet
trade (Costa & P
erez-Miles 2007). As other theraphosids,
this species is large sized, has low vagility and is long-lived,
with a lifespan of more than 30 years (Criscuolo et al.
2010). Females and juveniles are burrowers, while the
males are the more vagile sex, dispersing in search of
females during the mating season. Grammostola anthracina
has two known allopatric colour morphs: black populations
in the north of Uruguay and brown-reddish in the south of
the country, both apparently separated by the Negro river;
however, Postiglioni & Costa (2006) suggested that the iso-
lation of both morphs is due to ecological factors. Previous
studies have shown behavioural differences between these
colour morphs in copulatory duration and insertion pat-
terns (Costa & P
erez-Miles 2002; Postiglioni & Costa
2006). Although both morphs do not occur in sympatry,
when individuals of each morph are experimentally place
together, they can copulate. Moreover, they can mate with
G. iheringi, another species that occurs in a restricted area
of Uruguay. Notwithstanding, differences in distribution,
sexual behaviour and colour between the two morphs sug-
gest that they may be different species. First, to test the
hypothesis that G. anthracina as currently understood
(hereafter G. anthracina s.l.)comprises more than one spe-
cies, we rst conducted an exhaustive analysis of the distri-
bution of each of the morphs to investigate for patterns or
correlations with spatial variables. Second, we performed a
molecular analysis based on the cytochrome oxidase I
(COI) gene to determine the degree of genetic and
genealogical differentiation of both morphs; this gene has
been successfully used in spiders to elucidate the status of
cryptic species (e.g. Starret & Hedin 2007; Bond & Stock-
man 2008; Aguilera et al. 2009; Hamilton et al. 2014). We
used the genealogy herein as a guide to delimit and identify
candidate species. Finally, in the light of the genealogical
results, we use a process of reciprocal illumination
to search for diagnostic characters by re-examining the
external morphology of both morphs. As such, this contri-
bution provides a rst attempt to evaluate the taxonomy of
Grammostola using an integrative approach that employs
molecular and morphologic data; based on the gathered
results, we formally describe the species Grammostola quiro-
gai n. sp.
Materials and methods
Morph geographic distribution
We studied specimens housed in the Entomology Collec-
tion of the Facultad de Ciencias, Universidad de la
Republica, Uruguay, and specimens kept alive in the Ethol-
ogy, Ecology and Evolution Laboratory, of the Instituto de
Investigaciones Biol
ogicas Clemente Estable, Uruguay. We
also performed eldwork; we explored 26 hilly-rocky sites
in Uruguay during the years 20112013 and collected 65
specimens (22 and 43 brown-reddish and black individuals,
respectively) at 17 sites (Table 1). All specimens were
deposited in the Entomology Collection of Facultad de
Ciencias (FCE-MY), Universidad de la Republica, pre-
served in ethanol. Collection localities for each individual
were georeferenced, to generate a spatial distributional map
with the software DIVA-GIS (Hijmans et al. 2005) including
an ecoregion layer for Uruguay (Panario 1988). To test an
association between distribution and ecoregions, a logistic
regression was carried out using the package PAST (Paleon-
tological Statistics version 2.05, Hammer et al. 2001).
DNA-based analyses
Sampling design. All specimens collected by us (Table 1)
were sequenced. We also sequenced one specimen of
G. andreleetzi and G. burzaquensis from Uruguay, six Gram-
mostola pulchra de Mello-Leit~
ao 1921 from Brazil, and ve
Grammostola rosea (Walckenaer 1837) from Chile. All speci-
mens, preserved in ethanol, were deposited in the Ento-
mology Collection of Facultad de Ciencias (FCE-MY),
Universidad de la Republica. The ingroup was completed
with available GenBank haplotypes of specimens belonging
to the subfamilies Theraphosinae (Aphonopelma seemanni
(Pickard-Cambridge 1897): JN0181241; Eupalaestrus wei-
jemberghi (Thorell 1894): JQ412446) and Selenocosmiinae
(Chilobrachys huahini Schmidt & Huber 1996: JN018125;
Coremiocnemis cunicularia (Simon 1892): JN018198). Trees
were rooted with haplotypes of specimens of the subfamily
Ornithoctoninae: Cyriopagopus schioedtei (Thorell 1891)
(JN0181126) and Haplopelma schmidti von Wirth 1991
(JN018127 and AY309259). As such, the analysed matrix
comprises a total of 86 sequences.
DNA isolation, PCR and sequencing. Muscle tissue was
extracted from the right leg IV of each spider, removing
25 mg of tissue and stored in 100% ethanol at 80 °C.
Total genomic DNA was isolated using the DNeasyTissue
2ª2015 Royal Swedish Academy of Sciences
Species delimitation in the genus Grammostola L. Montes de Oca et al.
Table 1 List of collection localities of G. anthracina s.l. (see also Fig. 3). The ID represents the abbreviation for each locality
# Locality Locality ID Latitude Longitude Morph FCE-MY Accession Nos.
1 Masoller, Artigas Mas001 31.092 56.016 Black 0947 KT965200
1 Masoller, Artigas Mas002 31.092 56.016 Black 0948 KT965275
1 Masoller, Artigas Mas007 31.092 56.016 Black 0953 KT965201
2 Sepulturas, Artigas Sep094 30.830 56.054 Black 1246 KT965229
2 Sepulturas, Artigas Sep095 30.830 56.054 Black 1242 KT965234
2 Sepulturas, Artigas Sep096 30.830 56.054 Black 1245 KT965233
2 Sepulturas, Artigas Sep097 30.830 56.054 Black 1244 KT965230
2 Sepulturas, Artigas Sep098 30.830 56.054 Black 1243 KT965231
3 Arroyo Catal
an, Artigas Art064 30.816 56.350 Black 1204 KT965235
4 Baygorria, R
ıo Negro Bay035 32.864 56.827 Black 0935 KT965254
4 Baygorria, R
ıo Negro Bay036 32.864 56.827 Black 0936 KT965198
4 Baygorria, R
ıo Negro Bay037 32.864 56.827 Black 0937 KT965253
4 Baygorria, R
ıo Negro Bay038 32.864 56.827 Black 0938 KT965274
4 Baygorria, R
ıo Negro Bay040 32.864 56.827 Black 0940 KT965269
5 Cuchilla de Navarro, R
ıo Negro RNe061 32.666 57.026 Black 1235 KT965270
5 Cuchilla de Navarro, R
ıo Negro RNe062 32.666 57.026 Black 1203 KT965271
6 Cuchilla Negra, Rivera CNe076 31.074 55.976 Black 1227 KT965266
6 Cuchilla Negra, Rivera CNe081 31.074 55.976 Black 1230 KT965276
6 Cuchilla Negra, Rivera CNe075 30.951 55.626 Black 1008 KT965242
6 Cuchilla Negra, Rivera CNe077 30.951 55.626 Black 1231 KT965244
6 Cuchilla Negra, Rivera CNe078 30.951 55.626 Black 1229 KT965240
6 Cuchilla Negra, Rivera CNe079 30.951 55.626 Black 1228 KT965243
6 Cuchilla Negra, Rivera CNe080 30.951 55.626 Black 1226 KT965241
7 Arerungu
a, Salto Are058 31.462 56.714 Black 1212 KT965251
7 Arerungu
a, Salto Are093 31.462 56.714 Black 1199 KT965272
8 Daym
an, Salto Day051 31.401 57.691 Black 1215 KT965256
8 Daym
an, Salto Day052 31.401 57.691 Black 1232 KT965205
8 Daym
an, Salto Day053 31.401 57.691 Black 1201 KT965221
9 Achar, Tacuaremb
o Ach011 32.399 56.116 Black 0912 KT965239
9 Achar, Tacuaremb
o Ach012 32.399 56.116 Black 0915 KT965212
9 Achar, Tacuaremb
o Ach013 32.399 56.116 Black 0910 KT965264
9 Achar, Tacuaremb
o Ach014 32.399 56.116 Black 0914 KT965265
9 Achar, Tacuaremb
o Ach015 32.399 56.116 Black 0920 KT965273
9 Achar, Tacuaremb
o Ach016 32.399 56.116 Black 0913 KT965203
9 Achar, Tacuaremb
o Ach017 32.399 56.116 Black 0955 KT965268
10 Valle Ed
en, Tacuaremb
o VaE022 31.817 56.167 Black 0944 KT965214
10 Valle Ed
en, Tacuaremb
o VaE023 31.817 56.167 Black 0945 KT965252
10 Valle Ed
en, Tacuaremb
o VaE024 31.817 56.167 Black 0946 KT965216
10 Valle Ed
en, Tacuaremb
o VaE025 31.817 56.167 Black 0925 KT965215
10 Valle Ed
en, Tacuaremb
o VaE026 31.817 56.167 Black 0924 KT965213
11 Arerungu
a, Tacuaremb
o Are054 31.644 56.306 Black 1205 KT965204
11 Arerungu
a, Tacuaremb
o Are055 31.644 56.306 Black 1200 KT965199
11 Arerungu
a, Tacuaremb
o Are057 31.644 56.306 Black 1377 KT965217
12 Isla Patrulla, Treinta y Tres TyT084 33.059 54.543 Brown-reddish 1202 KT965223
12 Isla Patrulla, Treinta y Tres TyT085 33.059 54.543 Brown-reddish 1218 KT965224
12 Isla Patrulla, Treinta y Tres TyT086 33.059 54.543 Brown-reddish 1208 KT965225
12 Isla Patrulla, Treinta y Tres TyT087 33.059 54.543 Brown-reddish 1213 KT965227
12 Isla Patrulla, Treinta y Tres TyT088 33.059 54.543 Brown-reddish 1234 KT965228
13 Cerros de San Juan, Colonia CSJ041 34.187 57.927 Brown-reddish 0956 KT965247
13 Cerros de San Juan, Colonia CSJ042 34.187 57.927 Brown-reddish 0922 KT965202
13 Cerros de San Juan, Colonia CSJ043 34.187 57.927 Brown-reddish 0923 KT965250
13 Cerros de San Juan, Colonia CSJ044 34.187 57.927 Brown-reddish 0921 KT965245
13 Cerros de San Juan, Colonia CSJ046 34.187 57.927 Brown-reddish 0918 KT965238
13 Cerros de San Juan, Colonia CSJ047 34.187 57.927 Brown-reddish 0917 KT965255
14 Nueva Palmira, Colonia NPa074 33.850 58.412 Brown-reddish 1240 KT965246
15 Grutas del Palacio, Flores Flo082 33.849 56.970 Brown-reddish 1219 KT965232
15 Grutas del Palacio, Flores Flo083 33.849 56.970 Brown-reddish 1223 KT965248
ª2015 Royal Swedish Academy of Sciences 3
L. Montes de Oca et al. Species delimitation in the genus Grammostola
morphs are not sister to each other. However, given that
there are several known causes (including the fact that the
mitochondrial genome is only inherited by females) for
which a gene tree may depart from the species tree (Pamilo
& Nei 1988), the analysis of nuclear genes is needed to test
the obtained topology. Nonetheless, the phenome may be
considered as a good proxy of the nuclear genome because
most characters have genetic basis. Both morphs of G. an-
thracina s.l. not only differ from each other in coloration,
but also in leg morphology (Fig. 4) and behaviour. As such,
it is the integration of the genetic, phylogenetic and phe-
notypic results, including behavioural differences (Costa &
P
erez-Miles 2002; Postiglioni & Costa 2006) which sug-
gests that G. anthracina as currently understood is com-
posed of two species; one corresponding to the black
morph and other to the brown-reddish morph. Even when
the black morph and the brown-reddish morph clades show
internal structure, most of these subclades lack signicant
support and, more importantly, there are not morphologic
differences among them. As such, we consider these inter-
nal clades represent intraspecic variation. Therefore, we
consider further splitting of G. anthracina s.l. is not war-
ranted at the light of current data. Nonetheless, we
acknowledge that the direct assessment of the variation of
nuclear loci represents a step needed to further test our
taxonomic hypothesis.
The type locality of G. anthracina is in Montevideo,
Uruguay; even though the original description does not
consider the colour and tibial apophysis characters, we
can infer that it belongs to the brown-reddish morph
according to the distribution of this morph. As such, we
restrict the name G. anthracina to the brown-reddish
morph and describe the black morph as a new species
below:
Genus Grammostola Simon, 1892
Grammostola quirogai n. sp. (Figs 4B, 5AC, 6A,B)
Fig. 3 Network of haplotypes of the COI gene of Grammostola
anthracina s.s. (orange and red) and G. quirogai n. sp. (brown,
green and blue). Locality numbers as those of Table 1.
Fig. 4 Males right tibial apophysis on leg
I. A. Grammostola anthracina s.s. B.
Grammostola quirogai n. sp. C.
Grammostola iheringi.D. Grammostola
burzaquensis.E. Grammostola pulchra.
F. Grammostola rosea.
ª2015 Royal Swedish Academy of Sciences 7
L. Montes de Oca et al. Species delimitation in the genus Grammostola
tion, extending towards the north by the Uruguay River
coast and along the Santa Ana Sierra (Fig. 1). Both morphs
were recorded nearby at two areas. In the area of Queguay,
Paysandu in north-western Uruguay, they were recorded to
be 14 km from each other. The brown-reddish record is
based on a specimen collected in 2010 (Route 3 and Que-
guay River); in spite of an intense effort in the eld, we
could not nd other specimens of this morph in that area,
but we did nd a black individual in a locality (Route 26
near Araujo Stream) 14 km away. The second area where
both morphs were found is the conuence of the Santa
Ana Sierra and Negra Sierra. The black morph is on
Negra Sierra, 7 km apart from the Puntas del Cu~
napiru,
Santa Ana Sierra locality, where a specimen of the brown-
reddish morph was collected in 2001. This latter locality
has been modied by human activity, and during our eld
work, we did not nd any specimens. Association analyses
indicated a signicant relationship of both morphs with
distinct Uruguayan ecoregions. The black morph associates
with the Basaltic basin, whereas the brown-reddish morph
is found in the Laguna Merin formation, Santa Luc
ıa for-
mation and Eastern Sierras (v
2
=49.37, P<0.01). Gond-
wana and West sedimentary basins shared both morphs
(Fig. 1).
DNA-based results
Population and genetic divergence analyses. The analysed
matrix consisted of 568 bp, with 225 variable sites, of
which 181 were parsimoniously informative, and includes
50 distinct haplotypes. The sample of 65 sequences of
G. anthracina s.l. shows a high haplotypic diversity
(Hd =0.979; 42 distinct haplotypes). Nucleotide diversity
for the sample of G. anthracina s.l is 0.067, whereas the
sample of the brown-reddish and black morphs is 0.053
and 0.014, respectively. Average genetic divergence
between black and brown-reddish morphs is 10.3%. For
interspecic comparisons, the minimum genetic distance is
10% for the pair brown-reddish G. anthracina and
G. burzaquensis, and the maximum value, 14.6%, corre-
sponding to the comparison of G. pulchra (Brazil) and
G. andreleetzi (Uruguay).
From the 42 haplotypes of G. anthracina s.l., 28 were
recovered from specimens of the black morph and 14 from
individuals of the brown-reddish morph. An AMOVA indi-
cated that 56.23% of the total variation observed in G. an-
thracina s.l. is due to differences between morphs
(F
st
=0.86, P<0.001). Both morphs show considerable
geographic structure; in most cases, each locality studied
was represented by a unique haplotype not shared with
other populations. Intrapopulation genetic differentiation
was lower in the brown-reddish morph (0.33%) than in the
black morph (2.3%). Pairwise distances between localities
were low when comparing localities of the same morph
(brown-reddish: 0.017 0.009; black: 0.054 0.2).
Phylogenetic analysis. The topologies derived from the
three phylogenetic analyses (MP: consensus of 64 shortest
trees of length: 1718, CI: 32, RI: 79; log-likelihood:
3605.08) are highly congruent; one important discrepancy
is noted below. Therefore, only the Bayesian-derived
topology is shown (Fig. 2). Grammostola was recovered as
monophyletic (JK <50, BML: 0.69, PP: 0.99), but G. an-
thracina s.l. was not recovered as monophyletic. Haplotypes
of the black morph of G. anthracina form a clade (JK <50,
BML: 0.56, PP: 0.96) as well as those of the brown-reddish
morph (JK: 99, BML: 0.59, PP: 0.82), which are most sur-
prisingly not sister lineages. The black morph clade was
sister (JK <50, BML: 0.62, PP: 0.96) to G. pulchra. In the
MP and BI analyses, the brown-reddish morph of G. an-
thracina was recovered as sister (JK <50, PP: 0.82) to
G. burzaquensis. Meanwhile, in the ML tree G. burzaquensis
was recovered as sister (BML: 0.57) to the clade black
morph of G. anthracina and G. pulchra. Within the black
morph, clade haplotypes form three groups, which are not
allopatric. Meanwhile, the brown-reddish shows geographic
structure with haplotypes falling into two clades that are
geographically segregated, one in the west (localities:
Fig. 1 Distribution in Uruguay of the morphs of Grammostola
anthracina. Yellow circles show the areas where black and brown-
reddish morphs become closer. Delimitation of ecoregions,
according to the dominant type of rock, follows Panario (1988).
ª2015 Royal Swedish Academy of Sciences 5
L. Montes de Oca et al. Species delimitation in the genus Grammostola
Nueva Palmira, Cerros de San Juan and Estancia el
Timote) and another towards the east (localities: Pan de
Azucar, Isla Patrulla and Cuchilla de Navarro); the same
structure is also seen in the haplotype network (Fig. 3).
Morphological characters
Male tibial apophyses of the studied species differ in the
presence and distribution of spines and setae on the prolat-
eral (PB) and retrolateral (RB) branches. The brown-red-
dish morph has on the PB and RB a group of subapical
macrosetae, a mega-spine on the inner side of RB and an
apical retrolateral spine on tibia I (Fig. 4A), whereas the
black morph and the other studied species lack the group
of subapical macrosetae and each have a small spine on the
inner side of RB and 02 apical retrolateral spine on tibia I
(Fig. 4B). In addition, G. iheringi has a spine on the inner
side of PB and an apical retrolateral mega-spine on tibia I
(Fig. 4C). Males of G. burzaquensis have a spine on the
inner side of RB, and two apical retrolateral spines on tibia
I (Fig. 4D). Grammostola pulchra has on the RB a subapical
short spine and a spine in its inner side, a group of subapi-
cal macrosetae in PB, and a long apical retrolateral spine
on tibia I (Fig. 4E). Grammostola rosea alternatively has a
spine on the inner side of PB, and 12 long apical retrolat-
eral spines on tibia I (Fig. 4F). All species have a short api-
cal spine on RB (Fig. 4AF).
Taxonomic description
The mitochondrial data show that both morphs of G. an-
thracina s.l. are genealogical distinct (i.e. each one is mono-
phyletic) and that on average their haplotypes diverge by a
value (10.3%) that falls within the range of values observed
for all other species pairs of Grammostola (Table 2). In
addition, the mitochondrial gene tree shows that both
Fig. 2 Genealogical relationships among
haplotypes of the mitochondrial COI gene
of Grammostola as recovered in the
Bayesian analysis. Support for selected
nodes is indicated as follows: Jackknife
Bootstrap Posterior probability.
Non-signicant supported nodes are
without data. Terminal labels correspond
to localities as indicated in Table 1.
6ª2015 Royal Swedish Academy of Sciences
Species delimitation in the genus Grammostola L. Montes de Oca et al.
morphs are not sister to each other. However, given that
there are several known causes (including the fact that the
mitochondrial genome is only inherited by females) for
which a gene tree may depart from the species tree (Pamilo
& Nei 1988), the analysis of nuclear genes is needed to test
the obtained topology. Nonetheless, the phenome may be
considered as a good proxy of the nuclear genome because
most characters have genetic basis. Both morphs of G. an-
thracina s.l. not only differ from each other in coloration,
but also in leg morphology (Fig. 4) and behaviour. As such,
it is the integration of the genetic, phylogenetic and phe-
notypic results, including behavioural differences (Costa &
P
erez-Miles 2002; Postiglioni & Costa 2006) which sug-
gests that G. anthracina as currently understood is com-
posed of two species; one corresponding to the black
morph and other to the brown-reddish morph. Even when
the black morph and the brown-reddish morph clades show
internal structure, most of these subclades lack signicant
support and, more importantly, there are not morphologic
differences among them. As such, we consider these inter-
nal clades represent intraspecic variation. Therefore, we
consider further splitting of G. anthracina s.l. is not war-
ranted at the light of current data. Nonetheless, we
acknowledge that the direct assessment of the variation of
nuclear loci represents a step needed to further test our
taxonomic hypothesis.
The type locality of G. anthracina is in Montevideo,
Uruguay; even though the original description does not
consider the colour and tibial apophysis characters, we
can infer that it belongs to the brown-reddish morph
according to the distribution of this morph. As such, we
restrict the name G. anthracina to the brown-reddish
morph and describe the black morph as a new species
below:
Genus Grammostola Simon, 1892
Grammostola quirogai n. sp. (Figs 4B, 5AC, 6A,B)
Fig. 3 Network of haplotypes of the COI gene of Grammostola
anthracina s.s. (orange and red) and G. quirogai n. sp. (brown,
green and blue). Locality numbers as those of Table 1.
Fig. 4 Males right tibial apophysis on leg
I. A. Grammostola anthracina s.s. B.
Grammostola quirogai n. sp. C.
Grammostola iheringi.D. Grammostola
burzaquensis.E. Grammostola pulchra.
F. Grammostola rosea.
ª2015 Royal Swedish Academy of Sciences 7
L. Montes de Oca et al. Species delimitation in the genus Grammostola
Holotype. m, URUGUAY, Cuchilla del Daym
an, Route
31 km 24, 31°24002S, 57°41029W, 14 February 2013, F.
Costa, F. P
erez-Miles & L. Montes de Oca (FCE-MY
1215).
Paratypes. Artigas, Subida de Pena (1m FCE-MY 0195, 1f
FCE-MY 1249), Rivera, Camino a Portones Negros (1m
FCE-MY 1261, 1f FCE-MY 1264), Salto, Route 31 km 24
(1f FCE-MY 1201), Tacuaremb
o, Pozo Hondo-Tambores
(1m FCE-MY 0163, 2ff FCE-MY 0167), Tacuaremb
o,
Route 43 km 18 (1m FCE-MY 0626).
Etymology. The specic epithet is a patronym dedicated
to the memory of Horacio Quiroga, an Uruguayan poet,
playwright and master of short story writing, born in Salto
where the new species has been registered.
Diagnosis. This species can be distinguished from con-
geners by its dark coloration with some white-grey hairs.
Male differs from those of other species by the spine com-
bination of the tibial apophysis of leg I, having a small
spine on the inner side of RB, and 02 apical retrolateral
spine on tibia I.
Description
Male (holotype). Total body length, 39.24. Carapace
length 19.02, width 18.14. Anterior eye row recurve, poste-
rior procurve. Eyes sizes and interdistances: AME 0.25,
ALE 0.5, PME 0.47, PLE 0.5, AMEAME 0.5, AME
ALE 0.375, PMEPME 0.95, PMEPLE 0.175, ALEPLE
0.275, OQ length 1.325, width 2.175, clypeus 0.275. Fovea
transverse, straight, width 2.98. Labium length 1.985, width
2.58 with 144 cuspules, maxillae with 248/252 cuspules in a
Table 2 Intra- and interspecic distances (p-distance) for species of the genus Grammostola
Genetic distances between groups
G. quirogai
n. sp.
G. anthracina G. rosea G. pulchra G. burzaquensis
G. anthracina
0.103 ––
G. rosea
0.113 0.116 –– –
G. pulchra
0.102 0.107 0.112 ––
G. burzaquensis
0.109 0.1 0.126 0.105
G. andreleetzi
0.137 0.146 0.129 0.15 0.135
Genetic distances within groups 0.0554 0.013 0.0129 0.0431
Fig. 5 Grammostola quirogai n. sp. A.
Male, habitus. B. Left palpal bulb,
retrolateral. C. Left palpal bulb,
prolateral.
Fig. 6 Grammostola quirogai n. sp. A.
Female, habitus. B. Spermatechae,
dorsal view.
8ª2015 Royal Swedish Academy of Sciences
Species delimitation in the genus Grammostola L. Montes de Oca et al.
triangular group with base on the proximal edge. Sternum
length 7.4, width 6.5. Posterior sigillae submarginal.Che-
licerae with 11 promarginal teeth (second basal shorter)
and 4 little retromarginal. Tarsi I-IV densely copulate,
scopula entire. Metatarsi I-II completely scopulate, III
scopula on apical third, IV absent. Tibia I with paired dis-
tal proventral apophyses. Present one apical spine on the
retrolateral branch, one spine at the basis of the proventral
branch, and 02 retrolateral tibial spines (Fig. 4B). Flexion
of metatarsus retrolateral to tibial apophyses. Palpal organ
pyriform, curved and long embolus (Fig. 5B,C). Length of
leg and palpal segments, in Table 3. Spination: Femora:
palp 1P, I-IV 0. Patela: palp 1-1P, I-II 0, III 1P, IV 1R.
Tibia: palp 2-1 P, 1V; I 1-1 P, 1-1 R; II1-1-1-2 V, 1-1 R;
III 1-1 P, 1.1 V, 1-1-1 R; IV 1-1-2 D, 1-1 P. Metatarsus:
I1 V; II 2 V; III 2-1-1 P, 1-1-2 V, 1-1-1-2 R; IV 1-1-2 P,
1-1-1-2 V, 3R. Tibia: palp and I-IV 0. Colour:
Cephalothorax, abdomen and legs black with some grey
hairs (proximal to moult, the spider lost the intensity and
become brownish) (Fig. 5A). Type III-IV urticating hairs
present. PMS monoarticulated, PLS triarticulated, apical
segment digitiform.
Variation (range (mean standard deviation)): Total
length 39.251.2 (42.56 4.97), carapace length 19.223.3
(21.18 1.48), width 18.423.1 (20.76 1.93). AME
0.250.38 (0.32 0.05), ALE 0.50.64 (0.57 0.05),
PME 0.380.47 (0.39 0.04), PLE 0.480.68
(0.55 0.08), AMEAME 0.500.63 (0.59 0.06), AME
ALE 0.30.43 (0.38 0.05), PMEPME 0.971.88
(1.34 0.35), PMEPLE 0.130.3 (0.22 0.07), ALE
PLE 0.280.40 (0.33 0.06), OQ length 1.332.50
(2.04 0.45), width 2.182.8 (2.59 0.26), clypeus 00.63
(0.26 0.24). Fovea width 1.83.1 (2.66 0.55). Labium
length 1.72.8 (2.26 0.45), width 2.283.38
(3.05 0.46). Sternum length 7.49.1 (8.54 0.69), width
6.17.5 (6.94 0.62). Legs I 56.1464.1 (60.23 3.44), II
21.9527.6 (25.09 2.24), III 58.857.8 (53.6 3.72), IV
58.270.8 (64.46 5.03), palp 27.0831.77 (29.69 2.38).
Female (paratype FCE-MY: 1201). Total body length
46.21, carapace length 20.94, width 19.73. Anterior eye
row recurve, posterior procurve. Eyes sizes and interdis-
tances: AME 0.325, ALE 0.8, PME 0.5, PLE 0.575, AME
AME 0.6, AMEALE 0.25, PMEPME 1.25, PMEPLE
0.225, ALEPLE 0.275, OQ length 2.32, width 2.56,
clypeus 0.925. Fovea transverse, straight, width 5.2. Labium
length 2.65, width 3.14 with 117 cuspules, maxillae with
224/209 cuspules. Sternum length 8.27, width 8.75. Che-
licerae with nine promarginal teeth (from distal to proximal,
decreasing in size and the 8 is smaller than other); three retro-
marginal. Tarsi I-IV and palp densely scopulate, entire. Scop-
ula on metatarsi I 3/4, II 2/3, III 1/3 apical, IV absent. Length
of legs and palpal segments in Table 3. Spination: Femur:
palp 1P; I 1P; II-IV 0. Patella: palp and I-IV 0. Tibiae: palp 1-
1-3V, 1R; I 1-1-2V, II 1-1-1P, 1-1-2V, III 1-1-1P, 1-2V,
1-1R; IV 1P, 1-1V, 1-1R. Metatarsus: I 1-1-1V; II 1-3V; III 1-
1-1P, 1-1-2V, 1-1R; IV 1P, 1-1-1-1-1-1-2V, 1-1-1-1-1R.
Tarsus palp and I-IV 0. Colour: as in male (Fig. 6A). Type
III-IV urticating hairs present. PMS mono-articulated, PML
triarticulated, apical segment digitiform. Two straight
spermathecal receptacles (Fig. 6B).
Variation (range [mean standard deviation]): Total
length 36.752.8 (47.2 6.26), carapace length 17.423.27
(20.28 2.34), width 16.921.5 (19.8 2.01). AME 0.28
0.45 (0.35 0.07), ALE 0.480.8 (0.61 0.13), PME
0.280.5 (0.34 0.1), PLE 0.40.58 (0.5 0.08), AME
AME 0.550.75 (0.65 0.08), AMEALE 0.250.48
(0.35 0.09), PMEPME 0.971.5 (1.29 0.22), PME
PLE 0.130.26 (0.2 0.05), ALEPLE 0.250.4
(0.54 0.3), OQ length 1.882.9 (2.31 0.4), width 2.17
2.8 (2.53 0.23), clypeus 0.250.93 (0.54 0.3). Fovea
width 3.35.2 (3.91 0.76). Labium length 1.93.40
(2.7 0.59), width 3.13.3 (3.17 0.08). Sternum length
7.259.25 (8.26 0.78), width 5.88.77 (7.71 1.16).
Legs I 40.5653.36 (49.22 5.22), II 14.822.3
(18.85 2.74), III 33.149 (43 6.27), IV 41.858.6
(52.98 6.51), palp 25.0533 (30.64 3.22).
Discussion
Grammostola taxonomy has been problematic due to the
morphological homogeneity of its species, which is
reected in a number of critical changes of species compo-
sition over time (Bucherl 1951; Schiapelli & Gerschman
1961; World Spider Catalog 2015). The integration of
morphological, behavioural and molecular characters, con-
sidered together with distributional data, provides evidence
that has allowed us to uncover a new species herein
described as G. quirogai. This result shows the power of
integrating multiple lines of evidence in taxonomic studies.
Previous studies using DNA barcoding in Theraphosidae
have shown an average of 6% of divergence between
Table 3 Length (mm) of legs and palpal segments of the holotype
and one female paratype of Grammostola quirogai n. sp.
Femur Patella Tibia Metatarsus Tarsus
Male (FCE-MY 1215) palp 11.14 5.44 8 2.5
I 16.28 8.57 12.1 11.13 8.06
II 15.54 8.39 11.28 10.54 7.28
III 13.71 7.75 10.71 12.4 7.03
IV 16.39 7.81 13.05 15.75 7.79
Female (FCE-MY 1201) palp 11.68 6.82 7.27 6.5
I 15.41 9.75 11.64 9.13 6.93
II 14.41 8.51 9.94 8.8 6.1
III 12.36 7.7 8.18 9.55 5.81
IV 15.52 8.11 11.05 13.96 6.03
ª2015 Royal Swedish Academy of Sciences 9
L. Montes de Oca et al. Species delimitation in the genus Grammostola
congeneric species (Hamilton et al. 2011). Ours compar-
isons of species of Grammostola range between 10% and
15%. Morphological evidence found in spines and setae
from the male tibial apophysis as well as coloration also
support the discrimination among all the Grammostola spe-
cies, including G. anthracina s.s. and G. quirogai n. sp.
Grammostola anthracina shows low level of genetic varia-
tion; meanwhile, the genetic diversity seen in G. quirogai n.
sp is larger. In fact, the sample of G. quirogai n. sp is the
most variable of those here analysed (Table 2). This species
shows a relatively deep genealogy, but which is not geo-
graphically structured; clades show a large degree of geo-
graphic overlap, including localities were pairs of clades are
found in sympatry. The average observed variation between
the internal clades of G. quirogai n. sp (6.87.2%) is much
lower than of those values corresponding to comparisons
between species pairs of Grammostola (1014.3%; Table 2).
Given these results and the fact that those clades do not
differ morphologically, we treat them as representing
within species variation. However, we acknowledge that
until nuclear DNA sequences are analysed, we cannot com-
pletely rule out that those clades represent in fact distinct
isomorphic species. Having say that, if our taxonomic sce-
nario (i.e. G. quirogai n. sp is a species with relatively deep
mitochondrial genealogical structure) probes to be correct,
it would be of interest to understand the reasons why this
species appears at the mitochondrial level more variable
than its congeners so far analysed. Under a scenario of
neutrality, distinct demographic and historical scenarios
could produce the observed pattern (Avise 2000; see also
Lefer et al. 2012).
After exhaustive sampling, we did not nd G. anthracina
s.s. and G. quirogai n. sp. coexisting at the same locality.
Postiglioni & Costa (2006) suggested that the absence of
overlapping is mainly due to ecological factors. We also
consider that usually theraphosids have poor vagility (Fer-
retti et al. 2014) limiting the gene ow. The reported areas
of closer occurrence (Fig. 1) likely represent the distribu-
tional limits of each taxon; in these areas, individuals occur
in low density and are difcult to nd. Our present data
are not sufcient enough to determine whether these dis-
tributional ranges are stable or whether one species is dis-
placing the other.
Finally, our analyses also highlight additional areas of
study worth exploring. For example, most relationships
among species of Grammostola are poorly supported; as
such, the addition of taxa missing in our sampling together
with the analysis of nuclear DNA sequences is required to
more effectively investigate phylogenetic relationships of
these spiders. Such study is desirable before advancing a
scenario of historical biogeography accounting for the
diversication of Grammostola.
Acknowledgments
This research is the result of Laura Montes de Oca Master
in Science thesis. Her thesis was supported by PEDECIBA,
ANII and CSIC (Uruguay), Vogelspinnen I.G. Stuttgart &
Deutsche Arachnologische Gesellschaft e.V. (Germany).
Fernando Costa, Luc
ıa Ziegler Flavio Pazos, Nicol
as boul-
losan, Marcelo Loureiro, Sebasti
an Serra, Jos
e Bessonart,
Sebasti
an Fierro, Carolina Abud and Paco Majic provided
eld assistance. Mariana Cosse and Ivanna Tomasco helped
in the laboratory. Cecilia da Silva, Luc
ıa Ziegler, Lorena
Cohelo, Andr
es Parada, Alejandro D0Anatro and Leticia
Bidegaray provided assistance with the analyses. The
manuscript was greatly improved through the comments
provided by Dr. Jason Bond and two anonymous reviewers.
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12 ª2015 Royal Swedish Academy of Sciences
Species delimitation in the genus Grammostola L. Montes de Oca et al.
... However, the systematic scenario of this group is chaotic and problematic (Raven 1985) due to many factors: i) the morphology is usually conservative and exhibits a high level of homoplasy caused by low interspecific and high intraspecific variability (Pérez-Miles et al., 1996;Bertani 2000;Hamilton et al., 2016;Ortiz & Francke 2017); ii) the morphological delimitation is based on sexual characters and, therefore, sexually mature specimens are necessarily required, but some tarantulas take more than five years to become sexually mature and many are long-lived (Baerg 1938;Schwerdt et al., 2021); iii) most of the currently valid species and genera were described in the late 1800s, and most of these are poorly diagnosed or based on morphological characters that have proven not to be appropriate to define taxa; and iv) many holotypes of these species either are not associated with a specific type locality, are based on specimens that are not sexually mature, or specimens are lost or destroyed, have been badly preserved. Fortunately, the robustness of tarantula systematics considerable increased during the last years from many studies involving molecular systematics at genus and subfamilies levels (Hamilton et al., 2011;2014;Hendrixson et al., 2013;Wilson et al., 2013;Montes de Oca et al., 2016;Ortiz & Francke 2016;Mendoza & Francke 2017;2020;Lüddecke et al., 2018;Turner et al., 2018;Hüsser 2018;Fabiano-da-Silva et al., 2019;Foley et al., 2019;Candia-Ramírez & Francke 2020;Korba et al., 2023;Ferretti et al., 2023;Galleti-Lima et al., 2023). These studies have demonstrated that some molecular markers are useful to assess the evolutionary patterns of tarantula populations revealing cryptic or pseudo cryptic species, redundantly described taxa and intraspecific/interspecific paraphyly (Hamilton et al., 2016). ...
... In addition, because of the maternal inheritance of this marker, gene flow may also go unrecognized in organisms with male-mediated dispersal, such as mygalomorph spiders Satler et al., 2013;Ortiz & Francke 2016). Despite these limitations, COI sequences have proven to be useful to provide a framework for the taxonomy of some poorly studied groups, like tarantulas (Hamilton et al., 2011(Hamilton et al., , 2014(Hamilton et al., , 2016Hendrixson et al., 2013Hendrixson et al., , 2015Wilson et al., 2013;Graham et al., 2015;Hendrixson 2019;Ortiz & Francke 2014Longhorn et al., 2007;Petersen et al., 2007;Mendoza & Francke 2017;Montes de Oca et al., 2016;Candia-Ramírez & Francke 2020, Korba et al., 2023. ...
... Due to their homogeneity and conservative morphology, its taxonomy was in a chaotic state (Raven, 1990;Pérez-Miles et al., 1996;Bertani, 2001;Ferretti & Barneche, 2013). However, in the last two decades, theraphosid systematics has progressed considerably, recently the incorporation of molecular characters led several authors to propose well supported phylogenies (Hamilton et al., 2011(Hamilton et al., , 2014(Hamilton et al., , 2016Hendrixson et al., 2013Hendrixson et al., , 2015Wilson et al., 2013;Montes de Oca et al., 2016;Ortiz & Francke, 2016Mendoza & Francke, 2017, 2020Turner et al., 2018;Lüddecke et al., 2018;Hüsser, 2018;Fabiano-da-Silva et al., 2019;Foley et al., 2019Foley et al., , 2021Candia-Ramírez & Francke, 2020;Korba et al., 2022;Galleti-Lima et al., 2023;Biswas et al., 2023). Despite these advances, the diversity of tarantulas, mainly in the New World, probably remains underestimated but molecular markers have demonstrated to be useful tools to reveal cryptic species, redundantly described taxa, and intraspecific and interspecific paraphyly (Hamilton et al., 2016). ...
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We describe and illustrate a new tarantula species of the genus Plesiopelma from Lavalleja and Maldonado, Uruguay. Plesiopelma arevaloae sp. nov. is distinguished from other known species by morphological characters and molecular evidence.
... In this context, the combination of molecular and morphological data have been successfully 2 • Gardini et al. employed in recent years, proving to be useful in solving systematic issues in various taxa (e.g. Grismer et al. 2013, Montes de Oca et al. 2015, Liu et al. 2021, Li et al. 2022. ...
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Defining species boundaries may result challenging and has been a widely debated issue in the last decades. In cases of cryptic or "pseudocryptic" species, molecular approaches can be a valuable tool to provide taxonomic hypotheses and significantly complement morphological taxonomy. Here, two mitochondrial and one nuclear markers were used to study the phylogenetic relationships within the genus Tiroloscia, paying particular attention to Tiroloscia exigua. Moreover, we used a molecular clock to define a time window for the diversification of the main lineages within the species and explore aspects of its evolutionary history. Finally, four species delimitation methods were applied to clarify taxonomy and validate species boundaries. We found strong evidence against the monophyly of Tiroloscia and a surprisingly high level of genetic diversity within Tiroloscia exigua, supported by morphology. Notably, five evolutionary lineages were identified within T. exigua, suggesting the presence of distinct taxonomic entities. Divergence time estimation places the onset of T. exigua diversification around the middle Miocene (~12.2 Mya). Based on phylogenetic and morphological results, we propose the resurrection of Tiroloscia squamuligera as a valid species. Our results underscore the importance of molecular approaches to uncover hidden diversity, particularly in terrestrial isopods which may hide still underestimated biodiversity.
... To the extent that this has been measured, this expectation generally holds true. Strong genetic structuring has been measured in taxa from several continents (e.g., Bond et al. 2001;Arnedo and Ferrández 2007;Stockman and Bond 2007;Hamilton, Formanowicz, and Bond 2011;Hedin, Starrett, and Hayashi 2012;Opatova and Arnedo 2014;Opatova, Bond, and Arnedo 2016;Castalanelli et al. 2014;Harvey et al. 2015;Montes de Oca, D'Elía, and Pérez-Miles 2016;Starrett et al. 2018). Despite this extensive history of study, knowledge gaps remain. ...
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Although patterns of population genomic variation are well-studied in animals, there remains room for studies that focus on non-model taxa with unique biologies. Here we characterise and attempt to explain such patterns in mygalomorph spiders, which are generally sedentary, often occur as spatially clustered demes and show remarkable longevity. Genome-wide single nucleotide polymorphism (SNP) data were collected for 500 individuals across a phylogenetically representative sample of taxa. We inferred genetic populations within focal taxa using a phylogenetically informed clustering approach, and characterised patterns of diversity and differentiation within- and among these genetic populations, respectively. Using phylogenetic comparative methods we asked whether geographical range sizes and ecomorphological variables (behavioural niche and body size) significantly explain patterns of diversity and differentiation. Specifically, we predicted higher genetic diversity in genetic populations with larger geographical ranges, and in small-bodied taxa. We also predicted greater genetic differentiation in small-bodied taxa, and in burrowing taxa. We recovered several significant predictors of genetic diversity, but not genetic differentiation. However, we found generally high differentiation across genetic populations for all focal taxa, and a consistent signal for isolation-by-distance irrespective of behavioural niche or body size. We hypothesise that high population genetic structuring, likely reflecting combined dispersal limitation and microhabitat specificity, is a shared trait for all mygalomorphs. Few studies have found ubiquitous genetic structuring for an entire ancient and species-rich animal clade.
... This trend of incorporating DNA quickly diminishes, with no new species described using DNA from 2020 to 2022. There are only a handful of studies that have incorporated molecular data into their species delimitation studies and generic revisions including: Aphonopelma (Hendrixson et al., 2013;Hamilton et al., 2011Hamilton et al., , 2014Hamilton et al., , 2016, the Australian species (Briggs et al., 2023), Bonnetina (Ortiz and Franke 2015;2016, 2017, Brachypelma Simon, 1891 (Mendoza and, Davus O.Pickard-Cambridge, 1892 (Candia-Ramí rez and Francke, 2021), Grammostola Simon, 1892(Montes de Oca et al., 2016, Ischnocolus Ausserer, 1871(Korba et al., 2022, Pamphobeteus Pocock, 1901(Cifuentes et al., 2016, Plesiopelma Pocock, 1901(Ferretti et al., 2024, Tliltocatl Mendoza and Francke, 2020 (Mendoza and Francke, 2020), Lasiocyano, Parvicarina, Tekoapora (Galleti-Lima et al., 2023), and Urupelma (Kaderka et al., 2023). ...
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Systematics provides the foundational knowledge about the units of biodiversity, i.e., species, and how we classify them. The results of this discipline extend across Biology and can have important impacts on conservation. Here we review the systematic and taxonomic practices within Theraphosidae over the last 260 years. We examine the rate of newly described species and investigate the contemporary practices being used in the description of new genera and species. There have been two large waves of theraphosid taxonomy, with an explosive growth of newly described species and author combinations in the last 60 years. We look back and find that during 2010–2024 contemporary practices in theraphosid systematics and taxonomy have remained largely static, being dominated by morphology-based approaches. Over this period, only 10% of newly described species incorporated DNA data or explicitly stated the species concept used. Similarly for genera, only five of the 37 newly described genera over that time were supported as distinct and monophyletic by DNA. We highlight the taxonomic movement of species among Theraphosidae, Barychelidae, and Paratropididae; however, given the limited molecular sampling for the two latter families, the boundaries of these families remain a significant area of needed research. To promote inclusivity, we provide a copy of this paper in Spanish as supplementary material.
... As a consequence, repeated patterns of both morphological stasis and homoplasy are present among distantly related taxa (Hedin et al., 2019;Opatova et al., 2020;Wilson et al., 2023). Morphological homogeneity is particularly common at shallow phylogenetic level, but at the same time, mygalomorphs often possess a remarkably deep intra-specific genetic structuring (Bond et al., 2001) -Ramírez & Francke, 2021;Hamilton et al., 2011;Hedin, 2015;Leavitt et al., 2015;Montes de Oca et al., 2016;Newton et al., 2020;Satler et al., 2013). ...
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The outcome of species delimitation depends on many factors, including conceptual framework, study design, data availability, methodology employed and subjective decision making. Obtaining sufficient taxon sampling in endangered or rare taxa might be difficult, particularly when non‐lethal tissue collection cannot be utilized. The need to avoid overexploitation of the natural populations may thus limit methodological framework available for downstream data analyses and bias the results. We test species boundaries in rare North American trapdoor spider genus Cyclocosmia Ausserer (1871) inhabiting the Southern Coastal Plain biodiversity hotspot with the use of genomic data and two multispecies coalescent model methods. We evaluate the performance of each methodology within a limited sampling framework . To mitigate the risk of species over splitting, common in taxa with highly structured populations, we subsequently implement a species validation step via genealogical diversification index ( gdi ), which accounts for both genetic isolation and gene flow. We delimited eight geographically restricted lineages within sampled North American Cyclocosmia, suggesting that major river drainages in the region are likely barriers to dispersal. Our results suggest that utilizing BPP in the species discovery step might be a good option for datasets comprising hundreds of loci, but fewer individuals, which may be a common scenario for rare taxa. However, we also show that such results should be validated via gdi , in order to avoid over splitting.
... For some groups of arachnids, traditional morphology fails to recognize and delineate species boundaries. Also, identify sister or cryptic species requires other types or evidence such as molecular data, ecological niche modeling, morphometric morphology, haplotype networks, and biogeographical approximations (Hebert et al. 2003(Hebert et al. , 2004Hamilton et al. 2011Hamilton et al. , 2014Montes de Oca et al. 2015;Ortiz and Francke 2016;Cruz-López et al. 2019;Valdez-Mondragón et al. 2019;Newton et al. 2020;Valdez-Mondragón and Cortez-Roldán 2021). However, as demonstrated herein, spiders of the genus Physocyclus have robust morphology for diagnosis and identification at the species level, mainly of primary and secondary sexual characters, such as chelicerae and palps (males) or epigynes (females). ...
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Integrative taxonomy is crucial for discovery, recognition, and species delimitation, especially in underestimated species complex or cryptic species, by incorporating different sources of evidence to construct rigorous species hypotheses. The spider genus Physocyclus Simon, 1893 (Pholcidae, Arteminae) is composed of 37 species, mainly from North America. In this study, traditional morphology was compared with three DNA barcoding markers regarding their utility in species delimitation within the genus: 1) Cytochrome c Oxidase subunit 1 (CO1), 2) Internal Transcribed Spacer 2 (ITS2), and 3) Ribosomal large subunit (28S). The molecular species delimitation analyses were carried out using four methods under the corrected p -distances Neighbor-Joining (NJ) criteria: 1) Automatic Barcode Gap Discovery (ABGD), 2) Assemble Species by Automatic Partitioning (ASAP), 3) General Mixed Yule Coalescent model (GMYC), and 4) Bayesian Poisson Tree Processes (bPTP). The analyses incorporated 75 terminals from 22 putative species of Physocyclus . The average intraspecific genetic distance ( p -distance) was found to be < 2%, whereas the average interspecific genetic distance was 20.6%. The ABGD, ASAP, and GMYC methods were the most congruent, delimiting 26 or 27 species, while the bPTP method delimited 33 species. The use of traditional morphology for species delimitation was congruent with most molecular methods, with the male palp, male chelicerae, and female genitalia shown to be robust characters that support species-level identification. The barcoding with CO1 and 28S had better resolution for species delimitation in comparison with ITS2. The concatenated matrix and traditional morphology were found to be more robust and informative for species delimitation within Physocyclus .
... Much information about theraphosids, can be found both in classical (e.g., Simon 1864;Comstock 1980;Breene et al. 1996 among others) and in recent literature (Foelix 2011;Pérez-Miles 2020) of which, however, there are few published scientific works focused on life expectancy. It is demonstrated that tarantula species are very long-lived, with females that can live for 30 years or more (Costa & Pérez-Miles 2002;Criscuolo et al. 2010;Montes de Oca et al. 2016), always 3-4 years more than males that usually live until mating (Foelix 2011;Padilla et al. 2018). Males present a shorter lifespan compared to females (Pérez-Miles & Perafán 2020). ...
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Here we present the case of a male Chilean rose tarantula Grammostola rosea (Walckenaer, 1837) (Araneae: Theraphosidae) reared in captivity, whose life expectancy has been dated at 8.5 years. The specimen spent the last three as an adult. Both records are outside the average range of lifespan expected for an adult male. The specimen was kept under natural conditions in a Mediterranean climate, without photoperiod or temperature control. Diet was composed of Tenebrio molitor larvae and Gryllus sp./Acheta sp. nymphs and adults. The feeding regime was 1-2 prey per week in the early stages and later 1 prey every 20 days since the individual exceeded 15 mm body length approximately, to his death. The specimen spent most of its adult life without eating, actively refusing prey. The maximum time without eating was 22 months. At the time of death, the specimen measured 35 mm in body length and 130 with leg expanded. Previous research reported lower life expectancies in male tarantulas. More research on understudied lifecycle aspects of the Theraphosidae is needed.
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Tarantulas (Araneae: Theraphosidae) are one of the most diverse and widespread families of mygalomorph spiders, with over 1000 species recognised globally. While tarantulas can be found across most of mainland Australia, from arid regions to tropical forests, the Australian fauna are not yet well characterised. There are currently only 10 nominal species, up to 8 of which are currently recognised as distinct species. Here, we aim to undertake the first continent‐wide assessment of species diversity of tarantulas in Australia using an iterative, hypothesis‐testing approach. We apply a biological species concept and use DNA sequence data from three independent loci to delimit putative species based on evidence of lack of gene flow. First, we use the mitochondrial DNA marker 16S to identify a set of putative species hypotheses. We then test each hypothesis under the expectations of neotypy, allotypy and allophyly using two independent nuclear loci, EF1γ and 28S rRNA. Genealogically exclusive lineages are inferred using haplotype networks for each nuclear locus, interpreted to represent non‐interbreeding entities and hence represent distinct biological species. We find evidence for there being at least 20 distinct biological species of tarantula in Australia, with the highest species richness in northern Australia. Our results are in line with other DNA‐based studies of Australian mygalomorphs that have uncovered undescribed species diversity. Given the low number of samples included here, there is likely to be an even greater species diversity of tarantulas in Australia.
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New World tarantulas present a unique and conspicuous defen- sive mechanism: the release of urticating setae. The morpholog- ical differentiation of the types of setae suggests their distinct use, with two defensive mechanisms proposed: active defence against potential predators and passive defence against other arthropods, through the incorporation of setae into the moulting mat and/or ootheca. Tarantulas from Uruguay present three types of urticating setae with different morphologies (types I, III, and IV). It has been proposed that type I is used in passive defence and that type III in active defence; however, the use of type IV remains enigmatic. This study aims to elucidate the use of the type IV urticating setae. For this, we analysed oothecae of Grammostola anthracina, a species that presents type III and IV setae, quantifying them, and comparing the proportions between each type. Differences were found in the number of setae incor- porated into the ootheca: type IV urticating setae are present in a higher proportion compared to type III, which suggests their use mainly in passive defence. We also tested the effects of type IV urticating setae incorporated on oothecae on phorid larvae and ants.
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Abstract- Because they are designed to produced just one tree, neighbor-joining programs can obscure ambiguities in data. Ambiguities can be uncovered by resampling, but existing neighbor-joining programs may give misleading bootstrap frequencies because they do not suppress zero-length branches and/or are sensitive to the order of terminals in the data. A new procedure, parsimony jackknifing, overcomes these problems while running hundreds of times faster than existing programs for neighbor-joining bootstrapping. For analysis of large matrices, parsimony jackknifing is hundreds of thousands of times faster than extensive branch-swapping, yet is better able to screen out poorly-supported groups.
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The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
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Recent essays on the species problem have emphasized the commonality that many species concepts have with basic evolutionary theory. Although true, such consensus fails to address the nature of the ambiguity that is associated with species-related research. We argue that biologists who endure the species problem can benefit from a synthesis in which individual taxonomic species are used as hypotheses of evolutionary entities. We discuss two sources of species uncertainty: one that is a semantic confusion, and a second that is caused by the inherent uncertainty of evolutionary entities. The former can be dispelled with careful communication, whereas the latter is a conventional scientific uncertainty that can only be mitigated by research. This scientific uncertainty cannot be `solved' or stamped out, but neither need it be ignored or feared.