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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, 000–000.
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 insufficient. 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 difficult 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 difficult, if not impossible, task
(B€ucherl 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. B€ucherl 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 traffic 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 first 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 first 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 fieldwork; we explored 26 hilly-rocky sites
in Uruguay during the years 2011–2013 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 five
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 significant
support and, more importantly, there are not morphologic
differences among them. As such, we consider these inter-
nal clades represent intraspecific 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, 5A–C, 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 field, we
could not find other specimens of this morph in that area,
but we did find a black individual in a locality (Route 26
near Araujo Stream) 14 km away. The second area where
both morphs were found is the confluence 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 modified by human activity, and during our field
work, we did not find any specimens. Association analyses
indicated a significant 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
interspecific 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 0–2 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 1–2 long apical retrolat-
eral spines on tibia I (Fig. 4F). All species have a short api-
cal spine on RB (Fig. 4A–F).
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-significant 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 significant
support and, more importantly, there are not morphologic
differences among them. As such, we consider these inter-
nal clades represent intraspecific 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, 5A–C, 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°24002″S, 57°41029″W, 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 specific 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 0–2 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, AME–AME 0.5, AME–
ALE 0.375, PME–PME 0.95, PME–PLE 0.175, ALE–PLE
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 interspecific 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 0–2 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 39.2–51.2 (42.56 4.97), carapace length 19.2–23.3
(21.18 1.48), width 18.4–23.1 (20.76 1.93). AME
0.25–0.38 (0.32 0.05), ALE 0.5–0.64 (0.57 0.05),
PME 0.38–0.47 (0.39 0.04), PLE 0.48–0.68
(0.55 0.08), AME–AME 0.50–0.63 (0.59 0.06), AME–
ALE 0.3–0.43 (0.38 0.05), PME–PME 0.97–1.88
(1.34 0.35), PME–PLE 0.13–0.3 (0.22 0.07), ALE–
PLE 0.28–0.40 (0.33 0.06), OQ length 1.33–2.50
(2.04 0.45), width 2.18–2.8 (2.59 0.26), clypeus 0–0.63
(0.26 0.24). Fovea width 1.8–3.1 (2.66 0.55). Labium
length 1.7–2.8 (2.26 0.45), width 2.28–3.38
(3.05 0.46). Sternum length 7.4–9.1 (8.54 0.69), width
6.1–7.5 (6.94 0.62). Legs I 56.14–64.1 (60.23 3.44), II
21.95–27.6 (25.09 2.24), III 58.8–57.8 (53.6 3.72), IV
58.2–70.8 (64.46 5.03), palp 27.08–31.77 (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, AME–ALE 0.25, PME–PME 1.25, PME–PLE
0.225, ALE–PLE 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 36.7–52.8 (47.2 6.26), carapace length 17.4–23.27
(20.28 2.34), width 16.9–21.5 (19.8 2.01). AME 0.28–
0.45 (0.35 0.07), ALE 0.48–0.8 (0.61 0.13), PME
0.28–0.5 (0.34 0.1), PLE 0.4–0.58 (0.5 0.08), AME–
AME 0.55–0.75 (0.65 0.08), AME–ALE 0.25–0.48
(0.35 0.09), PME–PME 0.97–1.5 (1.29 0.22), PME–
PLE 0.13–0.26 (0.2 0.05), ALE–PLE 0.25–0.4
(0.54 0.3), OQ length 1.88–2.9 (2.31 0.4), width 2.17–
2.8 (2.53 0.23), clypeus 0.25–0.93 (0.54 0.3). Fovea
width 3.3–5.2 (3.91 0.76). Labium length 1.9–3.40
(2.7 0.59), width 3.1–3.3 (3.17 0.08). Sternum length
7.25–9.25 (8.26 0.78), width 5.8–8.77 (7.71 1.16).
Legs I 40.56–53.36 (49.22 5.22), II 14.8–22.3
(18.85 2.74), III 33.1–49 (43 6.27), IV 41.8–58.6
(52.98 6.51), palp 25.05–33 (30.64 3.22).
Discussion
Grammostola taxonomy has been problematic due to the
morphological homogeneity of its species, which is
reflected in a number of critical changes of species compo-
sition over time (B€ucherl 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 (6.8–7.2%) is much
lower than of those values corresponding to comparisons
between species pairs of Grammostola (10–14.3%; 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
Leffler et al. 2012).
After exhaustive sampling, we did not find 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 flow. 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 difficult to find. Our present data
are not sufficient 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
diversification 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
field 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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