Ecological analyses of the R. mexicanus species complex.
The partial ROC tests showed a significant predictive ability of the
models for all delimited species (p < 0.05; Appendix
7). ENMs predicted high-suitability areas for each of the delimited
species (Fig. 6). The R. mexicanus clade IIA had the largest
suitability areas, mainly distributed in the Sierra Madre Oriental,
northern Oaxaca, and the Central Highlands of Chiapas and Guatemala. For
the R. mexicanus clade I, suitability areas were found mostly in
the Sierra Madre Oriental and northern Oaxaca. The suitability areas ofR. mexicanus clade IIIA were restricted to the northern region of
the western and central Cordillera of the Colombian Andes, while theR. mexicanus clade IIIB occurs from the southwestern region of
the Colombian Andes to the northern Ecuadorian Andes.
There were significant pairwise differences between the delimited
species for all environmental variables except for Bio1 and Bio5 (Fig.
7a-g). The environmental niche of the R. mexicanus clade I
presented the lowest temperature values for the coldest month (Bio6,
mean of 6.5 °C). The R. mexicanus clade IIIA presented on average
the highest values of annual precipitation (Bio12, mean of 2483 mm) and
precipitation of the driest month (Bio14, mean of 95.2 mm) but showed
similar average values of precipitation of the wettest month, together
with the R. mexicanus clade IIA (Bio13, mean of 340.3 y 345.9 mm,
respectively). The CVA completely segregated the environmental niches ofR. mexicanus clade IIIA – R. mexicanus clade IIIB from
those of R. mexicanus clade I – R. mexicanus clade IIA,
but there was still a small region of overlap in their environmental
space (Fig 7h).
DISCUSSION
Reithrodontomys mexicanus cryptic species complex .
Reithrodontomys mexicanus originally comprised 13 subspecies
(Hooper 1952; Hooper 1955), though
10 are currently recognized (Bradley 2017). Our phylogenies included
samples of the formerly subspecies R. garichensis for the first
time. We confirmed that it is distinct from R. mexicanus but
belongs to the R. mexicanus group (Gardner and Carleton 2009).
Surprisingly, the Costa Rican specimens considered to beReithrodontomys sp. by Miller and Engstrom (2008) showed a close
relationship with R. garichensis . These authors suggested that
the two specimens from Volcan Poas, Costa Rica represent a new species
based on their morphological and genetic differences with respect toR. mexicanus . The three species delimitation methods implemented
here supported their proposal. On the other hand, these methods failed
to differentiate the specimen from La Carpentera, Costa Rica from that
of R. garichensis . Therefore, both were identified as the same
species, and the specimen from La Carpentera, Costa Rica is proposed to
be reclassified as R. garichensis .
In this study, R. mexicanus was recovered as a polyphyletic taxon
formed by three clades (I, II, III), highly divergent from each other,
which agrees with Arellano et al. (2003; 2005). Within clade II, all
delimitation methods demarcated individuals from Parque Nacional
Montecristo, El Salvador (clade IIB) to the species level. The cytb
genetic distance between this group and clade IIA was 4.96%, slightly
lower than the 5 % limit estimated by Baker and Bradley (2006) to
recognize sister mammal species. Specimens from El Salvador could not be
examined morphologically. Nonetheless, we had access to 5 individuals
from Los Esesmiles (Cerro El Pital); not included in the molecular or
morphometric analyses. Compared to individuals of R. mexicanusclade I distributed in Mexico and Guatemala, these specimens from Los
Esesmiles differ morphologically by their relatively shorter nasal
bones, broader palatal, and rounded braincase. The pelage exhibits a
cinnamon coloration, and the tail tends to be not much longer than the
head and body together. Parque Nacional Montecristo and Los Esesmiles
are only separated by ca. 30 km, so individuals from both localities
could be assumed to have the same genetic identity. Thus, populations
from El Salvador could be assumed to be a divergent lineage from the
remaining members of R. mexicanus clade I, but more evidence is
needed, such as molecular analyses that include an additional sampling
of individuals from this region, to reach a conclusion regarding their
taxonomic status.
The strongest morphometric similarities were found between the putative
species R. mexicanus clade IIA and R. mexicanus clade
IIIB, which showed no significant differences in the ventral skull
shape. It has been reported that the ventral view of the skull is the
one with a better phylogenetic signal (Camul and Polly 2005).
Consequently, our morphometric results are consistent with the molecular
data, which showed that these clades are more closely related to each
other than to the R. mexicanus clade I. In mammals, it has been
reported that the environment can influence the development of bone
structures such as the skull and jaw (Camul and Polly 2005). The few
morphometric differences found between these putative species (clades
IIA and IIIB) could be due to the fact that, in general, they share
similar habitat characteristics (Hooper 1952). However, the ecological
analyses allowed us to clearly segregate their environmental space,
based mainly on precipitation and VCF variables. Although their
separation was weakly supported by geometric morphometrics, the genetic
and environmental differentiation of these clades strongly support their
demarcation as distinct species.
The bGMYC delimitation method proved to be the most conservative for
proposing putative species within the clade that grouped the R.
mexicanus specimens from South America. Despite the number of species
proposed by the different methods, most pairwise comparisons did not
exceed cytb genetic distance values of 5% (K2P distances range from
1.50 to 4.20%, Supplementary material 2). Low levels of genetic
differentiation reported for South American rodents have been explained
as a consequence of recent speciation processes
(Patton and Smith 1992). Such
would be the case in the genus Reithrodontomys , whose
diversification processes began ca. 6.83 mya according to our results,
expanding from North America to South America, of which only subspecies
of R. mexicanus are known (Hooper 1952).
Arellano et al. (2005) analyzed a specimen from Colombia but retained it
as part of R. mexicanus sensu stricto. In their analyses,R. darienensis from Panama was not included. Our phylogenetic
analyses included R. darienensis and a good representation of
specimens from Colombia and Ecuador that had been classified a priori asR. mexicanus . Our evidence supported the conclusion that South
American specimens form a distinct clade from R. mexicanus which
is sister to R. darienensis . Within this clade, delimitation
methods and cytb genetic distances suggested that the Colombian
specimens (R. mexicanus clade IIIA) from Risaralda (ICN16579) and
Antioquia (FMNH78179) constitute a distinct species from the other South
American individuals. Although it was not possible to corroborate the
species-level distinction with morphological evidence, the ENM delimited
its distribution to the northwestern region of the western and central
Cordilleras of Colombia, with a habitat characterized mainly by high
precipitation values. These individuals could be considered a divergent
lineage that is already distinct from the rest of the South American
populations in at least three species properties (reciprocal monophyly,
genetic differentiation, and ecological niche distinctiveness; de
Queiroz 2007).
The remaining individuals from Colombia and Ecuador (R. mexicanusclade IIIB) were validated at the species level by genetic and
ecological data, and to a lesser extent with morphometric evidence.
Hooper (1952) reported three subspecies of R. mexicanus in South
America: R. m. milleri , R. m. soederstroemi , and R.
m. eremiscus . The known distribution of R. m. milleri ranges
from Colombia to northern Ecuador, including the suitability areas found
for the two candidate species (R. mexicanus clade IIIA andR. mexicanus clade IIIB). However, the distribution of R.
mexicanus clade IIIA was restricted to a small region of the western
and central Cordillera, while R. mexicanus clade IIIB was
distributed mainly in the Cordillera Oriental, a region not reported forR. m. milleri (Hooper 1952). In addition, the suitability areas
of R. mexicanus clade IIIB included localities recognized for the
other two subspecies (R. m. soederstroemi and R. m.
eremiscus ), distributed only in Ecuador (Hooper 1952; Arellano et al.
2015). Therefore, an analysis focused on the harvest mice populations of
South America is essential to correctly establish the taxonomic
designation of the Reithrodontomys species that inhabit this
region, since they undoubtedly do not belong to R. mexicanus .
Even though R.
mexicanus clade I had already been considered a candidate species using
allozymes (Arellano et al. 2003), cytb sequences (Arellano et al. 2005),
and chromosomal data (Urbina et
al. 2006), in our analyses, we were able to include a wide sampling that
allowed us to support this new species not only with molecular data but
also with morphological and ecological data. The phylogenetic position
of this putative species in the trees confirms that it is a much older
lineage (divergence time estimates ranging from 4.53 to 6.84 mya) and
has a different evolutionary history from the rest of the clades within
the subgenus Aporodon . Although representatives of the R.
mexicanus clade I have historically been classified as R.
mexicanus , they are genetically very distant from this species, even
those that coexist in sympatry in the localities of La Esperanza and
Puerto de la Soledad in Oaxaca, Mexico. Specimens of this clade could be
discriminated correctly by morphology, especially by ventral skull
shape. The phylogenetic signal that structures located on the ventral
side of the skull exhibit
(Lockwood et al. 2004; Macholán
2008) could explain the marked morphometric differentiation that this
clade presented in accordance with its position in the phylogenetic
trees. The environmental characteristics of this candidate species,
partially overlap with those of R. mexicanus clade II. This is
expected, given that they share part of their distribution in the
Mexican cloud forests (Gual-Díaz and Rendón-Correa 2014). However,
comparisons of most environmental variables were significantly
different, with R. mexicanus clade I occupying a geographic area
characterized by low values of temperatures and annual precipitation,
and high forest cover (VCF). The congruence between the independent
datasets is essential for the delimitation of this clade as a new
species since until now, only molecular data had been used to
differentiate it.
Taxonomy of the R. mexicanus species group .
Within the subgenus Aporodon , the R. mexicanus group
currently comprises the species R. mexicanus , R.
brevirostris , R. paradoxus , R. gracilis, R. spectabilis ,R. darienensis , and R. garichensis . The overall
distribution of this group ranges from Mexico to South America, although
most species are concentrated in Central America (Hall 1981). Here, all
the members of this group, but R. paradoxus , were analyzed using
molecular data, which allowed us to clarify the evolutionary
relationships between them and make taxonomic considerations within the
species group.
Reithrodontomys brevirostris was recovered as the sister group of
the R. mexicanus clade II and confirmed as a valid species by all
molecular delimitation methods. However, populations of this species
have tended to be confused with those of R. mexicanus from
Central America. Indeed, most of the individuals that exemplified theR. brevirostris clade in this study had originally been
identified by their collectors as R. mexicanus (and another two
as R. gracilis by Miller and Engstrom 2008). Similarly, in the
phylogeny of Arellano et al. (2005) one individual from Costa Rica,
grouped in their Clade I, was later reclassified as R.
brevirostris by Gardner and Carleton (2009). Furthermore, these later
authors assigned R. m. potrerograndei , a former R.
mexicanus subspecies, as part of R. brevirostris “because of
their comparably small size and other morphological resemblances”
(Gardner and Carleton 2009: 172). Hooper (1952) noted that many of the
morphological and cranial features of R. brevirostris were
reminiscent of R. mexicanus , but the absence of evidence of
interbreeding allowed them to be maintained as species. The separation
between R. brevirostris and R. mexicanus clade I occurred
at approximately 1.49 mya, and its genetic divergence for cytb was
5.68%, slightly higher than 5%, a generally observed distance between
sister species in mammals (Baker and Bradley 2006). The relatively low
genetic differentiation could account for the strong morphological
similarity historically reported between these clades. They also share
similar habitat characteristics, being distributed mainly in the cloud
forest (Hooper 1952, Gual-Díaz and Rendón-Correa 2014). Both clades
would fall within the gray zone described by de Queiroz (2007), in which
the decision as to whether they constitute one or two taxonomic entities
depends on the species criteria used. Based on our results, we propose
that they remain distinct entities, under the assumption that they have
been evolving as divergent lineages for sufficient time to separate but
continue to maintain many of the common ancestral characteristics they
share (de Queiroz 1998).
Hooper (1952) considered R. darienensis and R. gracilis to
be superspecies because they did not show major differences in
morphological traits, pelage coloration, or cranial or body size. The
term superspecies was proposed by
B. Rensch and later by E. Mayr to
refer to monophyletic and allopatric taxa that formed a single entity
and later evolved to the species level (Amadon 1966). However, our
phylogenetic results did not recover R. darienensis and R.
gracilis as a monophyletic group, and the genetic distances between
them reached values of almost 14%. Furthermore, R. darienensiswas more closely related to the clade containing R. mexicanusspecimens from South America (although they were genetically
well-differentiated) than to R. gracilis . This is consistent with
its restricted distribution in eastern Panama (Bradley 2017). TheR. gracilis specimens from Yucatan and Campeche, Mexico were
unequivocally delimited as the same entity as R. spectabilis ,
while those from El Salvador were recognized at the species level.
According to their geographical distribution, these two specimens
correspond to the subspecies R. g. anthonyi (Hall, 1981), but the
genetic distances of almost 8% between these and the R. gracilis+ R. spectabilis clade suggest that it is necessary to reevaluate
the Central American populations of R. gracilis , to assess if
they completed speciation processes
(Futuyma 2013).
Reithrodontomys spectabilis , whose distribution is restricted to
Cozumel Island, Mexico, was described as one of the largest species of
the genus (Jones and Lawor 1965). Although many aspects of its
morphology were reminiscent of R. gracilis from Yucatan, marked
differences in body size, darker coloration, and broader and heavier
zygomatic arches prompted its recognition at the species level. Jones
and Lawlor (1965) suggested that the precursor of R. spectabilisarrived from the Yucatan Peninsula during the Late Pleistocene, which
assumes a relatively long period of isolation between these two species.
Our results suggest that the divergence between these species (95% HPD
= 0.19-0.47) occurred at some point in the Middle Pleistocene (from
0.781 to 0.126 Mya, Walker et al.
2018), indicating a very recent separation between their populations
compared to those reported for other species of the subgenusAporodon (Martínez et al. 2022a; this study). This recent
separation is also consistent with the low cytb genetic differentiation
between these species (0.7 %), which fall within the intraspecific
range values proposed for Reithrodontomys (Baker and Bradley
2006). The phylogenetic relationships between R. spectabilis andR. gracilis have been analyzed in the past with allozymes and the
cytb gene, arriving at similar results to ours and suggesting an island
effect as a possible cause of their morphological differences
(Arellano et al. 2003; 2005).
Rodents frequently exhibit island gigantism with respect to conspecific
populations on the mainland
(Lomolino 1985). This phenomenon
is known as the Island rule
(Foster 1964) and is affected by
different factors including resources availability and the absence of
natural predators (Lomolino 2005).
We agree with Arellano et al. (2005) that this phenomenon could explain
why the harvest mice populations of Isla Cozumel differ, mainly in body
size, from the R. gracilis populations of Yucatan.
Recognizing R. spectabilis as a conspecific of R. gracilisentails reevaluating its populations in many ways, considering that it
is an endemic species classified as Critically Endangered by the IUCN
Red List of Threatened Species
(Vázquez et al. 2018). Additional
comparative studies of both species employing different sources of
evidence such as geometric morphometric, ecological niche, population
genetics, among others, are necessary to reach a conclusion about their
taxonomic status.
Species delimitation and its taxonomic implications .
Establishing species boundaries is difficult when dealing with
taxonomically complex groups whose descriptions have been based
primarily on their morphology (Dayrat 2005). Many of these species
exhibit such a pronounced morphological resemblance to each other that
they are recognized as cryptic species
(Bickford et al. 2007). This is
the case of R. mexicanus , where molecular (Arellano et al. 2003,
2005; Mill4er Engstrom 2008) and craniodental (Gardner and Carleton
2009) data have revealed broad cryptic variation leading to its
recognition as a species complex. Integrating multiple approaches to
delimit species has been strongly recommended to better confirm the
species hypothesis (Dayrat 2005;
Will et al. 2005). In this study,
we implemented an integrative taxonomy approach to test if there are
cryptic lineages within R. mexicanus that are evolving
separately. We employed different criteria in accordance with the GLC
(de Queiroz 1998; 2007), seeking as much evidence as possible to support
the recognition of identified candidate species
(Sangster 2018).
Species proposals were not always congruent among the delimitation
methods. The limitations of methods based on DNA data have been
discussed previously (see Luo et
al. 2018), mainly those related to non-compliance with the assumptions
of the method (Carsten et al. 2013). However, in Reithrodontomys ,
the efficacy of the delimitation methods used here (mPTP, bGMYC, and
STACEY) to demarcate cryptic lineages at the species-level has been
demonstrated (Martínez-Borrego et al. 2022a. The use of different
molecular markers showed conflicting results among the phylogenies, with
a high discordance between the topology of the cytb and those of the
Fgb-I7 and IRBP, respectively. This mito-nuclear discordance has been
suggested to be a consequence of introgression or incomplete lineage
sorting (Toews and Brelsford 2012;
Firneno et al. 2020). Although including more genetic markers can help
to elucidate species limits, sometimes the use of multiple loci has
complicated this purpose for taxonomists
(Firneno et al. 2021). Therefore,
in our study, the recognition of taxonomic entities was based primarily
on molecular species delimitation methods, including the cytb genetic
distances, traditionally used in mammal genetic studies (Bradley and
Baker 2001; Baker and Bradley 2006), but other evidence such as skull
morphometry and ecological niche were also used.
Geometric morphometrics and niche modeling have shown great
applicability in taxonomic studies in mammals
(Barčiová 2009; Martínez Gordillo
et al. 2010) and have allowed the corroboration of species limits
hypotheses derived from phylogenetic studies (e.g., Camul and Polly
2005; Rivera et al. 2018). However, the species-level clades proposed
here by the three delimitation methods and genetic distances were not
always strongly supported by ecological and/or morphological data. Under
the GLC, failure to meet any of the species criteria does not
necessarily mean that the candidate species does not correspond to a
divergent lineage (de Queiroz 2007). Rather, GLC recognizes that species
properties may evolve at different times during divergence (Sangster
2018), hence the importance of integrating multiple data sources to
support or reject the species hypothesis (Padial et al. 2010).
CONCLUSIONS
This work confirms that R. mexicanus sensu lato is a cryptic
species complex composed of at least four putative species: R.
mexicanus clade I, R. mexicanus clade IIA (R. mexicanussensu stricto), R. mexicanus clade IIIA, and R. mexicanusclade IIIB. In addition, specimens from El Salvador (R. mexicanusclade IIB) should be reevaluated taxonomically including a better
sampling of multiple lines of evidence. For R. mexicanus sensu
stricto, additional analyses are necessary to estimate its phylogenetic
relationship with respect to the subspecies R. mexicanus
riparius , which was not included in our analyses but has evident
geographic isolation from the other Mexican populations (Hooper 1955;
Hall 1981). Finally, R. mexicanus clade I constitute a new
species, pending formal description and assignment of a scientific name
according to the International Code of Zoological Nomenclature rules.
AUTHOR CONTRIBUTIONS
Daily Martínez-Borrego: Conceptualization (equal),
investigation (lead), data curation (lead), formal analysis (lead),
methodology (lead), writing original draft (lead), review and editing
(equal), visualization (lead). Elizabeth Arellano:Conceptualization (equal), methodology (supporting), writing – review
and editing (supporting), resources (lead), supervision (lead).Francisco X. González-Cozátl: Conceptualization (equal),
methodology (supporting), writing – review and editing (supporting),
supervision (supporting). Sandra M. Ospina-Garcés:Conceptualization (equal), methodology (supporting), writing – review
and editing (supporting). Duke S. Rogers: Conceptualization
(equal), methodology (supporting), writing – review and editing
(supporting), resources (supporting).
ACKNOWLEDGMENTS
We thank the following collections for providing samples (tissue or
destructive sample or specimens loans): Department of Mammalogy,
American Museum of Natural History (R. Voss); Colección Nacional de
Mamíferos, UNAM (F. A. Cervantes); Colección de Mamíferos de El Colegio
de la Frontera Sur, San Cristóbal (C. Lorenzo); Division of Mammals,
National Museum of Natural History (D. Lunde). The Field Museum of
Natural History (L. Heaney and A. W. Ferguson); Collection of Genetic
Resources, Genetic Resources Collection, Museum of Texas Tech University
(C. D. Phillips); Mammalogy, Royal Ontario Museum (M. D. Engstrom);
Museum of Zoology, Louisiana State University (D. Dittmann); Museum of
Zoology, University of Michigan (P. Tucker and C. Thompson); Museum of
Southwestern Biology (J. Cook and M. Campbell); Museo de Zoología
Alfonso L. Herrera, Facultad de Ciencias, UNAM (L. León-Paniagua);
Colección Mastozoológica del Instituto de Investigaciones Biológicas,
Universidad Veracruzana (A. González). Permits for fieldwork in Mexico
were issued by the Secretaría de Medio Ambiente y Recursos Naturales. We
thank the American Society of Mammologists, Consejo Nacional de Ciencia
y Tecnología (CONACYT), and Universidad Autónoma del Estado de Morelos
for the funding provided. We thank D. Cruz for his help with the map
drawing and N. Lewis-Rogers for her support in the laboratory work.
FUNDING INFORMATION
DM-B was supported by a Ph.D. Scholarship Program 2018-000012
01NACF-11852LANC from Consejo Nacional de Ciencia y Tecnología
(CONACYT), Mexico. Fieldwork season 2019-2020 was carried out thanks to
the Latin American Field Research Award 2019 from the American Society
of Mammologists.
CONFLICT OF INTEREST
The authors declare no competing interests.
DATA AVAILABILITY STATEMENT
- DNA sequences: GenBank accession numbers ON156861-ON156912 (Cytb),
ON156913-ON156967 (Fgb-I7), and ON156968-ON156971(IRBP).
- Parameter selection for the construction of ecological niche models:
FigShare doi:
10.6084/m9.figshare.22190878.
- Occurrence points and ecological data: FigShare doi:
10.6084/m9.figshare.22194610.
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Table 1. Partition schemes and evolutionary models used in the
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