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Molecular time estimates for the Lagomorpha diversification
Divergence times for the Lagomorpha
https://orcid.org/0009-0008-6839-0232
Iraçabal Leandro Conceptualization Data curation Formal analysis Investigation 1
Barbosa Matheus R. Data curation Formal analysis 1
Selvatti Alexandre Pedro Data curation Formal analysis Writing – review & editing 2 *
https://orcid.org/0000-0002-1252-9206
Russo Claudia Augusta de Moraes Conceptualization Funding acquisition Project administration Writing – review & editing 1 *
1 Departamento de Genética, Rio de Janeiro, Universidade Federal do Rio de Janeiro, CCS, Instituto de Biologia, Rio de Janeiro, Brazil
2 Departamento de Zoologia, Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcântara Gomes, Maracanã, Rio de Janeiro, Brazil
Janke Axel Editor
Senckenberg am Meer Deutsches Zentrum fur Marine Biodiversitatsforschung, GERMANY
Competing Interests: The authors have declared that no competing interests exist.

* E-mail: apselvatti@gmail.com (APS); claudiaamrusso@gmail.com (CAMR)
6 9 2024
2024
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© 2024 Iraçabal et al
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Iraçabal et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Despite their importance as members of the Glires group, lagomorph diversification processes have seldom been studied using molecular data. Notably, only a few phylogenetic studies have included most of the examined lagomorph lineages. Previous studies that included a larger sample of taxa and markers used nonconservative tests to support the branches of their proposed phylogeny. The objective of this study was to test the monophyly of families and genera of lagomorphs and to evaluate the group diversification process. To that end, this work expanded the sampling of markers and taxa in addition to implementing the bootstrap, a more rigorous statistical test to measure branch support; hence, a more robust phylogeny was recovered. Our supermatrix included five mitochondrial genes and 14 nuclear genes for eighty-eight taxa, including three rodent outgroups. Our maximum likelihood tree showed that all tested genera and both families, Leporidae and Ochotonidae, were recovered as monophyletic. In the Ochotona genus, the subgenera Conothoa and Pika, but not Ochotona, were recovered as monophyletic. Six calibration points based on fossils were used to construct a time tree. A calibration test was performed (via jackknife) by removing one calibration at a time and estimating divergence times for each set. The diversification of the main groups of lagomorphs indicated that the origin of the order’s crown group was dated from the beginning of the Palaeogene. Our diversification time estimates for Lagomorpha were compared with those for the largest mammalian order, i.e., rodent lineages in Muroidea. According to our time-resolved phylogenetic tree, the leporids underwent major radiation by evolving a completely new morphospace—larger bodies and an efficient locomotor system—that enabled them to cover wide foraging areas and outrun predators more easily than rodents and pikas.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Education Ministry of Brazil (CAPES) Finance Code 001 https://orcid.org/0009-0008-6839-0232
Iraçabal Leandro National Research and Technology Council (CNPq) 310567/2018-1 da Cunha Matheus Rubens Barbosa Rio de Janeiro State Research Funding Agency (FAPERJ) E-26/010.001887/2019, SEI-260003/001170/2020, SEI-260003/012995/2021 Selvatti Alexandre Pedro Rio de Janeiro State Research Funding Agency (FAPERJ) 277324 Selvatti Alexandre Pedro This study was financially supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Education Ministry of Brazil (CAPES) in the form of a grant (Finance Code 001) received by LI. This study was also financially supported by the National Research and Technology Council (CNPq) in the form of a grant (310567/2018-1) received by CAMR and a Master's fellowship award received by MRB. This study was also financially supported by Rio de Janeiro State Research Funding Agency (FAPERJ) in the form of grants (E-26/010.001887/2019, SEI-260003/001170/2020, SEI-260003/012995/2021) received by CAMR and a grant (E-26/203.840/2022) (277324) received by APS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are in public databases or within the manuscript and its Supporting Information files.
Data Availability

All relevant data are in public databases or within the manuscript and its Supporting Information files.
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pmcIntroduction

Rabbits, hares, and pikas are part of the mammalian order Lagomorpha. These herbivorous mammals have a highly modified jaw morphology that lacks canine teeth and includes gnawing-specialized sharp incisors that grow continuously [1]. These unique features are shared with their sister group, the species-rich large mammalian order Rodentia. Together, rodents and lagomorphs encompass the Glires grandorder, which belongs to the Euarchontoglires clade [2]. Unlike rodents, lagomorphs have one additional small pair of upper incisors that are located behind the larger pair common to all mammals [3].

There are approximately 90 extant species of Lagomorpha split into two living families, distinguishable by the presence of three molar pairs in the upper jaw in Leporidae and only two in Ochotonidae [1, 4]. The monogeneric Ochotonidae includes the living genus Ochotona, which is divided into 30 species of hamster-like pikas. They live in Central Asia, Japan and North America and are distinguished from Leporids by their relatively short ears, small hind feet, and lack of a conspicuous tail [5 p. 127]. The Leporidae family includes 10 genera and 60 species of rabbits and hares that are characterized by elongated ears and large hind feet [6].

Historically, the geographical distribution of the lagomorphs included all continents but not Oceania or any other islands. However, some European lagomorphs have been deliberately introduced in Australia, mostly for hunting purposes, as rabbits remind nostalgic European colonists of their faraway home [7]. Despite their importance, only a handful of studies have included global phylogenetic reconstruction and divergence time estimates on the Lagomorphs. Some phylogenetic studies have been conducted, but their analyses have been restricted to specific genera (Lepus: [8, 9]; Ochotona: [10–13]) or subgenera (Ochotona: [14]; Pika: [15–17]; Conothoa: [18]) and, therefore, have a limited taxonomic scope.

Other studies have included phylogenetic reconstruction with broader sampling but are still restricted to Leporidae [19–21] or Ochotonidae [22]. Averianov’s work, for instance, is an analysis of the phylogeny of 28 extant and extinct leporids based on 31 morphological characters. Unfortunately, morphologically based phylogenies are poorly resolved, as expected because of the low number of morphological characteristics used. On the other hand, the work of Matthee and collaborators [20] used 27 species of leporids with five nuclear and two mitochondrial markers. These authors analysed their data using several tree-building methods that yielded relatively high confidence values, particularly for Bayesian inference. For ochotonids, one of the most comprehensive works is that of Yu et al. [22], who used 23 species but included only two mitochondrial markers.

To date, the two major exceptions in terms of the taxonomic scope of lagomorphs are the work on diversification and biogeography [23] and the supertree by Stoner and collaborators [24]. The latter performed a supertree analysis, compiling phylogenies based on morphological and molecular data from 146 lagomorphic articles. Despite the importance of such comprehensive analyses, supertrees come with statistical problems related to the difficulties assessing the reliability of their branches, among other issues (see [25]).

Conversely, in the work of Ge and coworkers [2013], the authors used three mitochondrial markers and Bayesian inference to reconstruct the history of Lagomorph diversification, including a time tree with a detailed analysis of biogeography. However, the time priors (or time calibrations) used in that study were secondary calibrations that were converted into a single point age for Lagomorpha with no confidence interval. Simulated and empirical data have shown that secondary calibrations and the lack of confidence interval in point calibrations leads to uncertainty on divergence time estimates, especially when the node age is shifted from the true age with falsely high precision [26–28].

Divergence times based on multiple fossil calibrations distributed across the internal nodes tend to be more reliable, as this strategy incorporates good rate variation among branches and drastically reduces the errors from single fossil analyses [27, 29–31]. Thus, in the present work, the sampling of markers and species was expanded, totalling five mitochondrial and 14 nuclear genes and 80 ingroup species, to generate a time tree for Lagomorpha. We used multiple fossils as time priors, including the root and internal nodes of the tree according to best practices [32]; such calibrations were resampled to ascertain their robustness. In addition, the bootstrap test of support relies on ping, a more conservative and reliable statistical support method, was used to measure the support of the internal branches [33]. The objectives of the article are to detail the temporal diversification process of the order Lagomorpha using a more complete dataset, a more robust time tree and more rigorous statistical analyses than did previous studies.

Methods

The taxonomic reference of this paper was the Higher Taxonomy database [34] (S1 Table). We included the type species for eight genera, namely, Lepus (L. timidus), Nesolagus (N. netscheri), Ochotona (O. dauurica), Pentalagus (P. furnessi), Poelagus (P. marjorita), Pronolagus (P. crassicauatus), Romerolagus (Romerolagus diazi) and Sylvilagus (S. floridanus), as well for four Ochotona subgenera: Ochotona (Conothoa) roylii, Ochotona (Lagotona) pusilla, Ochotona (Ochotona) dauurica and Ochotona (Pika) alpina.

Our data matrix included all available DNA sequences in GenBank for Lagomorpha, and as outgroups, three Rodentia species were selected, for a total of eighty-eight taxa sampled from the GenBank database [35]. Five mitochondrial genes and 14 nuclear genes were included as markers. All access codes can be found in S2 Table. The alignment of each marker was performed in MAFFT v.7 [36]. The iterative refinement algorithm FFT-NS-i was used for all the markers, except for the ribosomal subunit 12S, for which the Q-INS-i algorithm was selected because it considers the secondary structure of the RNA. The other parameters were set to their defaults in both cases. The concatenation was performed with the AMAS tool [37], and the matrix was composed of 17,938 sites.

For phylogenetic reconstruction and branch support, the IQ-Tree v. 2.1.3 program [38] was used. The alignment was initially divided into 19 partitions, and the ModelFinder algorithm, as implemented in IQ-Tree, was used to calculate the best molecular evolution model for each partition [39]. The algorithm was also used to estimate the best alignment partition scheme, grouping partitions with the same evolutionary models and avoiding problems associated with overparameterization [40]. The criterion used to choose the models and the partition scheme was Bayesian information (BIC), as it is a more conservative estimate [41]. The final scheme consisted of nine partitions with six different models of sequence evolution.

To remove unstable lineages, 1,000 ultrafast bootstrap pseudoreplicates were used as input for RogueNaRok, a software package that identifies rogue taxa [42]. The algorithms used for identification were strict and majority-rule consensus to explore a greater number of potential rogues [33]. The analysis was performed with the parameter "max. dropset size" set at 1 to 4 for the strict consensus and at 1 and 2 for the majority-rule consensus, aiming to allow the grouping of up to four tips in the calculation of the improvement of the bootstrap value. The threshold used to define a lineage as a rogue was 2 for strict consensus and 0.5 for majority rule consensus. Therefore, Bunolagus monticularis, Lepus starcki, Sylvilagus andinus, Sylvilagus bachmani, and Sylvilagus cunicularis were removed, decreasing the number of species in the final matrix to 80 lagomorphs. Our analysis focused on the rogue-excluded maximum likelihood analysis bootstrap support, but the phylogenetic tree on which the rogues were kept is available in the Supplemental Material (S1 Fig). After rogue removal, the sequences were aligned, producing a final matrix of 17,942 sites, and the phylogeny was inferred using the cited protocols (Fig 1).

10.1371/journal.pone.0307380.g001 Fig 1 Phylogenetic relationships and divergence times for Lagomorpha using the maximum likelihood algorithm, 79 terminal taxa, five mitochondrial genes and 14 nuclear genes mitochondrial genes (17,942 sites).

The bootstrap values (1,000 ultrafast bootstrap pseudoreplicates) lower than 100 are shown. All species were considered as Rogue Taxa (Roguenarok program) were removed from the alignment. Timetree produced from a maximum likelihood phylogeny and six calibrations, with 95% confidence intervals shown as bars at nodes. Colors represent different epochs.

The divergence times were estimated using the RelTime method with the MEGA 11 program [43, 44] with fossil-based calibrations (Table 1). A problem commonly associated with non-Bayesian approaches for divergence time inference was once the impossibility of including the calibration data as a probability distribution, but this became possible with the RelTime program [45]. We used a lognormal distribution for all calibrated clades to include uncertainty about the maximum age of the fossils. Initially, seven calibration points previously used to date the lagomorph tree were considered. However, following best practices for the inclusion of fossils in molecular dating [32], only six samples were retained (Table 2). In this case, the calibration of Lepus was removed because it was extracted from an indirect estimation [46].

10.1371/journal.pone.0307380.t001 Table 1 Age constraints of six nodes.

Calibrated clade	Hard minimum age	Soft maximum age	Lognormal distribution parameters	References	
Glires	59.24	162.5	59.24, 2, 1.35	Li et al. (2016), Wang et al. (2016), He et al. (2022)	
Rodentia	56	66	56, 1, 0.67	Marivaux et al. (2004)	
Mus-Rattus	10.4	16	10.4, 0.8, 0.47	Benton et al. (2015)	
Lagomorpha	48.07	66	48.07, 2, 0.45	Rose et al. (2008)	
Leporidae	8	11.65	8, 0.5, 0.41	Flynn et al. (2014)	
Stem Sylvilagus	3	4.75	3, 0.3, 0.13	Gazin (1942), Panseko & Lucas (2011)	

10.1371/journal.pone.0307380.t002 Table 2 Time estimates for major Lagomorpha lineages using the seven calibration schemes.

	Lagomorpha	Leporidae	Ochotonidae	Lepus	Sylvilagus	Nesolagus	Pronolagus	
Calibration Nodes/Range	56.00–47.80	4.75–3	20.44–15.97	13.82–11.63	5.33–3.60	-	7.25–5.33	
All included	57.2	25.4	15.4	4.5	6.5	12.7	12.4	
Glires excluded	62.0	27.4	16.7	4.9	6.6	13.7	13.3	
Lagomorpha excluded	62.5	27.1	16.8	4.7	6.1	13.6	13.2	
Leporidae excluded	57.9	26.4	15.6	4.8	7.4	13.2	12.9	
Mus+Rat excluded	57.6	25.6	15.5	4.6	6.6	12.8	12.5	
Rodentia excluded	53.1	23.7	14.3	4.3	6.6	11.9	11.6	
Sylvilagus excluded	57.8	27.2	15.6	5.2	10.3	13.6	13.3	

To test the consistency and influence of each calibration point in the analysis, internal consistency tests were performed with several rounds of calibration point sets. In set 1, all six calibration points were included (Table 2), and in the remaining sets, each calibration point was removed in turn. The ages used in the analyses were based on the work of Gradstein et al. [47] on geologic timetables. When a more accurate or up-to-date age delimitation was found in the relevant literature, this was used (see Table 1, references therein).

Results

In our tree, the Lagomorpha order and the two Lagomorpha families, Leporidae and Ochotonidae, were found to be monophyletic and to have high bootstrap (BP) support values (100% BP for each) (Fig 1). Eight genera were included in our Leporidae clade. Among these, Lepus (30 spp.; 100 BP), Pronolagus (four species, 100 BP), Nesolagus (two species, 100 BP) and Sylvilagus (10 species, 100 BP) were recovered as monophyletic clusters, whereas Caprolagus, Pentalagus, Poelagus and Romerolagus, the only extant species, were included in our dataset. Among the ochotonids, Ochotona, the only extant genus, was also recovered in a tight cluster (29 species, 100 BP).

Leporidae

In our Leporidae tree (Fig 2), our small Leporidae clade (93 BP), containing the genera Nesolagus, Poelagus and Pronolagus, was found to be the sister of a larger Leporidae clade (45 species, 92 BP) that included the genera Caprolagus, Lepus, Pentalagus, Romerolagus and Sylvilagus (see also Cano-Sanchéz et al. 2022) (Fig 2). In the small clade, the monotypic Poelagus species joined (99 BP) the four-species cluster of Pronolagus (4 species 100 BP), in which P. crassicaudatus was a sister to P. randensis (94 BP) and P. rupestres clustered with P. saundersiae (100 BP). This lineage was a sister (100 BP) to the Nesolagus cluster (100 BP), which included N. netscheri plus N. timminsi.

10.1371/journal.pone.0307380.g002 Fig 2 Phylogenetic relationships and divergence times for Leporidae clade (pruned from Fig 1).

In the large Leporidae clade (Fig 2), the monotypic genus Romerolagus (R. diazi) was a sister to a highly supported group (93 BP) with Caprolagus, Lepus, Pentalagus and Sylvilagus. In sequence, the split was between the large genus Lepus (30 species, 100 BP) and the diversity that included the Sylvilagus clade (10 species, 100 BP) plus the group (2 species, 99 BP) with the monotypic Pentalagus and Caprolagus.

According to our tree (Fig 2), the Sylvilagus cluster is monophyletic (100 BP) and divided into two lineages. The smaller lineage (89 BP) included a two-pair set of species: S. aquaticus plus S. palustris (100 BP) and S. audubonii plus S. nuttallii (100 BP). In the larger Sylvilagus lineage (100 BP), S. brasiliensis joined S. dicei (100 BP), and they clustered with another set (83 BP) of two pairs: S. floridanus plus S. robustus (100 BP) and S. obscurus and S. transitionalis (100 BP).

Our Lepus tree included 30 species (Fig 2), approximately twice as many species as the most comprehensive molecular phylogenetic work to date [23]. In this lineage, the species Lepus sinensis was a sister of the remaining Lepus diversity (29 species, 66 BP), but support was low. In the main Lepus group, the split separates the large clade (21 species, 64 BP), which includes L. arcticus, L. capensis, L. castroviejoi, L. comus, L. coreanus, L. corsicanus, L. europaeus, L. granatensis, L. habessinicus, L. hainanus, L. mandshuricus, L. nigricollis, L. oiostolus, L. othus, L. peguensis, L. sinensis, L. tibetanus, L. timidus, L. tolai, L. townsendii and L. yarkandensis from the smaller clade (nine species, 53 BP) with L. alleni, L. americanus, L. brachyurus, L. californicus, L. callotis, L. flavigularis, L. microtis, L. saxatillis and L. schlumbergeri.

In the small Lepus clade (Fig 2), the first split separated L. brachyurus from the remaining species into two lineages. In the first lineage (99 BP), L. americanus was a sister of the group (100 BP) joining two clades: one is L. californicus plus L. alleni (97 BP), and the other is L. callotis plus L. flavigularis (50 BP). In the second (93 BP), L. schlumergeri with L. microtis (100 BP) was a sister to L. saxatilis.

In the large Lepus clade, two sister lineages are shown. In the first (53 BP), L. arcticus joined (83 BP) L. castroviejoi plus L. corsicanus (100 BP). This species was a sister to L. othus (98 BP), followed by L. timidus (68 BP) and L. townsendii (97 BP). This group was a sister to L. capensis and the L. mandshuricus and L. coreanos clade (100 BP). L. tolai was a sister of this nine-species group (89 BP), and the clade (96 BP) of L. yarkandensis and L. tibetanus diverged next. This diversity was like that of a group (82 BP) containing two clusters of three species each. In the first (86 BP), L. granatensis was a sister of the L. europaeus plus L. habessinicus clade (100 BP), and in the second (100 BP) clade, L. nigricollis was a sister of the L. hainanus and L. peguensis clade (98 BP, Fig 2).

Ochotonidae

Ochotona traditionally has been divided into four subgenera (Conothoa, Lagotona, Ochotona, and Pika) that are distinguished by the type of habitat or geographical region they occupy [11, 48]. A fifth subgenus, Alienauroa, was recently proposed based on genomic data [49, 50], and in our study, Ochotona syrinx was the sole representative of this subgenus. Lagomorphs of the subgenus Pika are ochotonids with a North American distribution, and the subgenus Ochotona includes species that live in steppes and shrub environments, whereas, in the subgenus Conothoa, we find species that inhabit mountains. The subgenus Lagotona was proposed to include a single species, O. pusilla [51].

As previously proposed [11], the subgenera Conothoa (100 BP) and Pika (100 BP) are both monophyletic according to our phylogeny. In our tree, Ochotona (Alienauroa) syrinx was loosely clustered (85 BP) with the clade (87 BP) that included Ochotona (Lagotona) pusilla and the species assigned to the subgenera Pika and Ochotona. O. iliensis, O. koslowi and O. vizier were not included in previous subgenus assignments but were tightly grouped (100 BP) in the Conothoa subgenus, whereas O. cansus and O. sikimaria were clustered (100 BP) with high bootstrap support in the Ochotona subgenus (Fig 1).

The Pika subgenus (100/100) includes North American ochotonids and is arranged as follows in our tree: The first divergence observed is between O. collaris + O. princeps (100 BP), followed by the divergence of O. alpina plus the O. turuchanensis clade (100 BP), O. argentata plus O. pallasi (100 BP), which was the sister of the remaining four species (98 BP), and O. hoffmanni as a sister of the polytomy (98 BP), O. hyperborea plus O. mantchurica plus O. coreana.

The shrub and steppe species are the ochotonids of the Ochotona clade (100 BP). In the present work, O. thomasi was recovered as a sister to the remaining subgenus (100 BP), followed sequentially by O. dauurica (99 BP), O. thibetana (99 BP), O. cansus (99 BP) and O. sikimaria, which is the sister of the final O. curzoniae + O. nubrica (100 BP) clade.

The species that inhabit the mountains make up the Conothoa clade (100 BP). In this group, the first split was between the O. erythrotis and O. forresti clades (93 bp), and the remaining diversity (100 bp) of the subgenus was observed. The next split included the O. vizier and O. rufescens clades (100 BP) and the remaining Conothoa diversity (80 BP). This diversity was divided into two lineages. The first (BP 89) O. ladecensis was a sister to O. koslowi plus O. iliensis (91 BP), whereas in the second (89 BP), O. rutila was a sister to O. roylii and the O. macrotis clade (89 BP).

Discussion

Lagomorpha time-resolved phylogenetic tree

The Lagomorphs first differentiated during the Palaeogene, while the Leporidae and Ochotonidae families started to differentiate in the upper Palaeogene and middle Neogene, respectively (Fig 1). The origin of Lepus dates from the Pliocene, while the age of Sylvilagus dates from the late Miocene. The internal consistency test of the calibrations detected differences between the time estimates when a specific calibration point was absent. However, in most calibration schemes, the ages were congruent. When there was a difference, usually 10% difference, the confidence intervals overlapped. Importantly, the age estimates for Lagomorpha and Leporidae remained stable when their specific calibration points were excluded. However, when the Rodentia calibration was excluded, the age of Lagomorpha recovered to the lower Eocene. Leporidae, on the other hand, originated in the upper Oligocene in every calibration scheme, while Ochotonidae was repeatedly found in the transition between the lower and middle Miocene.

The time tree with all calibration fossils indicated that the beginning of the crown group of lagomorphs was 57.2 million years ago (57.2–52.9 Ma), indicating that it appeared in the Thanetian Age, late Palaeocene, Palaeogene period (Table 2, Fig 1). The estimate by Ge et al. [23], which calibrated only the Lagomorpha node of the ingroup and with a single point in time (50.2 Ma), is younger (50.3 Ma) but consistent with our estimate. Notably, when the calibration for this node was not used in our study, the origin estimate was 62.52 Ma, increasing this difference slightly and with a wider confidence interval (74.8–52.2 Ma). The age proposed by Matthee et al. [20] of 29.0 (±3.8) Ma for Lagomorpha is considerably younger than our age, as these authors calibrated their tree with much younger ages (20–40 Ma) for the nodes. Nevertheless, their calibrations do not seem to correspond to the most recent fossil evidence, which indicates the existence of stem Leporidae and Ochotonidae since the Eocene [51, 52]. In addition, Matthee et al. [20] used a calibration for Lepus based on divergence time estimates and not on fossil occurrence. Cano-Sanchéz et al. [53] reported a Palaeocene age of ca. 60 Ma for Lagomorpha.

Among the two extant Lagomorpha clades, Leporidae was the first to diversify; it was dated to 25.4 Ma, the Chattian age, and the upper Oligocene. Ge et al. [23] found an age of approximately 18.1 Ma in the lower Miocene. Halanych and Robinson [54] used the average sequence distance between leporid genera and assumed a molecular clock to calculate the Leporidae divergence time; a slightly older age of approximately 12.2 to 16.3 Ma was found for this family. Similarly, Matthee et al. [20] reported an age of 14 (±1.5) Ma for this node. An even older age of 21.9 (± 3.8) Ma was found by Su and Nei [55] using Ig heavy chain variable region genes. The oldest estimated age for Leporidae was that proposed by Cano-Sanchéz et al. [53], with a divergence occurring ca. 47 Ma in the middle Palaeogene.

Ochotonidae, on the other hand, was recovered at 15.4 Ma in the Langhian, middle Miocene. Melo-Ferreira et al. [16] calibrated only the Lagomorpha node, testing three different ages, 31, 37 and 65 Ma, and found that the ages of Ochotonidae were 6.6, 7.8 and 13.8 Ma, respectively. Notably, no calibration points overlapped our prior distribution for this node (48–59 Ma). When Melo-Ferreira et al. [16] employed the oldest calibration (65 Ma), the age recovered was 13.8 Ma, the closest to the age estimated here. Similarly, Mohammadi et al. [54] used four calibration schemes to calibrate the Lagomorpha node, adding a 52 Ma calibration point to those previously used by Melo-Ferreira et al. [16]. The youngest age obtained was 5.7 million years ago, while the oldest was 12.5 Ma. Ge et al. [23] reported an age of 13.4 Ma for Ochotonidae, which is also the middle Miocene.

There is a clear pattern in the rich fossil record of lagomorphs; after the divergence of the Leporidae and Ochotonidae, the two families diverged quickly, producing a few stem lineages during the Eocene and the Oligocene [23, 51, 52]. The Leporidae family appeared in the fossil record at approximately 37 Ma, during the upper Eocene period, followed by Ochotonidae at approximately 33 Ma, during the Oligocene period [51]. At the Oligocene and Miocene boundaries, distinct patterns emerged in each lineage. The Ochotonidae ancestor experienced a burst of diversification at the earliest Miocene, centred in Asia, followed by range expansions to Europe, North America and Africa [51, 56]. Between the middle and late Miocene, a second wave of ochotonids that included the extant genus Ochotona replaced several primitive genera in Asia and other continents [51]. In contrast, the Leporidae diversity remained relatively low throughout the Miocene but radiated profusely by the latest Miocene [23, 51, 52]. Leporids have also expanded their ancestral range from Asia to Europe, Africa and North America since the late Miocene [23, 51]. Therefore, the rich Lagomorpha fossil record clearly indicates that Leporidae is older than Ochotonidae, but that the major diversification of Ochotonidae predated that of Leporidae.

However, such a clear-cut pattern supported by a rich fossil record on multiple continents does not fit the current hypothesis for lagomorph diversification [23]. For instance, in that study, the diversification of Leporidae predated that of the extant Ochotona radiation. Although that study dated the Ochotona clade to ~13 Ma, a fossil of an undetermined Ochotona species from the Amuwusu Micromammals Site (Miocene of China) extends the age of the genus to ~16 Ma [57]. Since fossil age uncertainty and internal calibrations were not incorporated in the Ge et al. [23] time tree, such a recent age for Ochotona might have been an artefact. In our time tree, we incorporated multiple calibration points and fossil age uncertainties as probabilistic distributions. Our age for the Ochotona ancestor was estimated to be 15.4 Ma, which better fits the minimum age for the genus in the fossil record, especially because the Ochotona fossil from the Amuwusu site was not included in our calibration set. Last, in the Ge and coworkers time tree, the diversification of crown Leporidae was dated to approximately 18 Ma. Since stem representatives of Leporidae first appeared 37 Ma, that scenario requires a 19 million-year [Myr] gap in the fossil record for stem Leporidae, which is otherwise very rich on several continents. Our age for crown Leporidae was estimated at 25.4 Ma with a small confidence interval, thereby reducing the fossil gap to only 12 Myr (Fig 2). Therefore, our time tree better fits the fossil record for both crown clades of Lagomorpha.

In the upper Neogene, adaptive radiation started in the lineage of the genus Lepus and the lineage of Sylvilagus. The large genus Lepus originated at approximately 4.5 Ma during the Zanclean stage. Matthee et al. [20] reported the origin of this genus in Zanclean, with an age of 5.2 (±0.5) Ma. Ge et al. [23], on the other hand, recovered an age of approximately 8.6 million years ago, shifting the origin of Lepus to the Miocene epoch. The most recent estimated age for Lepus was that proposed by Cano-Sanchéz et al. [53], with the most recent common ancestor dating to 2.5 Ma (95% CI: 1–4). Nesolagus diverged from Pronolagus and Poelagus by 18.4 Ma, and Pronolagus and Poelagus diverged 15 Ma in the middle Miocene. The beginning of the diversification of the clade comprising Pentalagus, Caprolagus and Sylvilagus began at approximately 14.6 Ma, with Sylvilagus separating it from the other genera at 7.8 Ma. The divergence of this clade from Sylvilagus occurred at the beginning of the Tortonian Age, with Sylvilagus appearing at approximately 6.5 Ma in the Messinian Age in the Miocene, in contrast with the age of 5.3 (±0.8) Ma reported by Matthee et al. (2004). The latter is much closer to the age recovered by Ge et al. (2013), with a Sylvilagus origin at 5.6 Ma. In addition, Cano-Sanchéz et al. [53] reported Sylvilagus as a paraphyletic taxon with respect to Brachylagus, and the age of this clade was ca. 1.5 Ma. The origin of the genus Ochotona was approximately 15.4 Ma, and its three subgenera had ages of 12.2 Ma (Conothoa), 11.8 Ma (Pika) and 6.1 Ma (Ochotona).

Among the seven time-resolved phylogenetic trees reconstructed in this study, there were some differences between the one that included all six fossils and the one in which one of the fossils was absent for the first lagomorph divergence (Table 2, Fig 1). This finding demonstrates the importance of including crucial calibration points. However, the congruence of most ages in the different calibration sets shows the robustness of the divergence time results of this part of the time tree.

The divergence time for crown Lagomorpha was consistent, approximately 58 million years ago, in almost all the sets, with only one exception. In the set without the Rodentia calibration point, there was a slight difference, with a younger age of 53 Ma. In the set without the calibration point of Lagomorpha itself, the oldest age is observed at approximately 62.5 Ma. Interestingly, the Leporidae family was consistent across all datasets, ranging from 23.7 to 27.4 Ma, and always recovered in the Chattian age. For Lagomorpha, when the Rodentia calibration was absent, the youngest age of 23.7 Ma was found. Ochotonidae showed the most stable age throughout the sets, at approximately 15.7 Ma, at the Langhian–Burdigalian boundary. The age of the Lepus genus was remarkably constant at 4.7 million years ago, always at the Zanclean age. The genus Sylvilagus, with a constant age of 6.7 Ma, differed in the set without the stem Sylvilagus calibration, with an age of 10.3 Ma.

Mammal and plant radiation

The current macroevolutionary explanation for the distinct diversification patterns in the lagomorph fossil record is also tentative. In the late Miocene, low atmospheric CO2 concentrations, seasonal fires and aridification fuelled grassland expansion worldwide, coinciding with the extinction of most ochotonids while leporids radiated [23]. That study suggested a direct link between C4 plant diversification, which predominates in the diet of leporids, and a decrease in the diversity of C3 plants, which are preferred by ochotonids (Fig 3). However, although C4 grasses are a major component of plant biomass in grasslands, recent studies have shown that many C3 grasses (e.g., Pooideae) also exhibited great diversity in the late Miocene [58]. Furthermore, other C3 plant families, such as daisies (Asteraceae), are often as diverse as (or more diverse than) grasses in grassland biomes [59]. Analyses of diversification rates and environmental variables have demonstrated that although grasses strongly differentiated during the late Miocene (~10 Ma), the same process was mirrored by the hyperdiverse Asteraceae [59]. Most importantly, surveys of food preference revealed Asteraceae as the dominant food preference in extant pikas [23, 60]. Additionally, the great diversity of Ochotonidae in the fossil record of the early Miocene closely matches the peak in diversity of Asteraceae within the same time frame ([59], Fig 3). Therefore, food preference alone does not explain the correlation between grassland expansion and the diversification–extinction patterns observed in Lagomorpha.

10.1371/journal.pone.0307380.g003 Fig 3 Diversification scenario of Lagomorpha and grasslands during the late Cenozoic.

As most studies in Lagomorpha have focused on comparisons between its internal lineages, the macroevolutionary relationships between lagomorphs and other small mammals remain to be explored. The species-rich rodents (order Rodentia) are the leading model for small mammals due to their sheer morphological, ecological and geographic diversity [61–63]. Most importantly, they are the sister group of Lagomorpha that forms the clade Glires, and divergence time estimates based on nuclear and mitochondrial data suggest that the burst of adaptive radiation in rodents occurred in the last 10 Ma [64]. As Rodentia and Lagomorpha share the characteristic of small body sizes and both have a pair of continually growing chisel-like incisors [61, 63], we suggest that such similarities in form and function are strong indicators of interspecific competition.

Therefore, to explain the diversity trends in Lagomorpha, we suggest a key biotic component that hinges on competition for resources with rodents. Fossils of stem Glires (e.g., Mimotonidae and Eurymylidae) indicate that small, rat-sized bodies and limb proportions are likely ancestral morphologies [61, 63, 65]. Palaeontological and molecular data agree that by the late Miocene, the Muroidea ancestor originated from the most diverse Rodentia families, Muridae and Cricetidae, which spread from Asia to other continents while consuming a variety of diets, from generalist omnivore diets (Muridae) to the strictly herbivorous diets consumed by Arvicolinae (Cricetidae) [62]. When this diversification pattern is compared with our chronological tree, we observed that the tempo and locale of the muroid radiation coincided with the drastic reduction in Ochotonidae genera in the fossil record (Fig 3).

Given that the two lineages share the ancestral Glires body plan and geographic ranges, we suggest that direct competition between members of the generalist and diverse Muroidea steered the mass extinction of the herbivorous Ochotonidae in the late Miocene. A recent study provided empirical evidence that rodent and pika populations interact enough to allow parasite spillover in North America [66]. An increase in rodent diversity has also been linked to extinction by competition with Multituberculata, a lineage closely related to the eutherian clade that disappeared when the major rodent lineages appeared in the fossil record worldwide [61, 63].

According to our hypothesis, while pikas competed for resources with muroid rodents of similar body sizes, the Leporidae ancestor would have escaped the turmoil of that competition by evolving a strikingly different body plan. Analyses of the complete fossil record of Glires in North America revealed a rise in Leporidae specialized in cursoriality (adapted to run) during the late Miocene [67]. Although Rodentia and Lagomorpha evolved burrowing habits and high crowned teeth (hypsodonty) suited to abrasive food such as that containing silica and dirt present in grasslands in the Miocene, only Leporidae showed increased cursorial specialization.

Leporidae species have overall larger body sizes than most Glires species [23, 68], and pikas and very few rodents exhibit true cursorial specializations due to their generally small size [68, 69]. Leporids, however, exhibit several unique adaptations to cursoriality, namely, a unique intracranial joint at the occipital region that alleviates spinal rebound during hopping [69] and extensive maxillary fenestration that provides mechanical resistance while reducing bone mass [70]. Although pikas exhibit some degree of maxillary fenestration, this characteristic became much more pronounced in the late Miocene Leporidae [69]. Finally, in the postcranial skeleton, the late Miocene Leporidae evolved elongated hindlimbs and metatarsals [68]. Therefore, the rapid diversification of the late Miocene Leporidae was marked by key innovations, such as powerful hindlimbs and cranium that withstand the mechanical pressures of high-speed locomotion. These are unique features of this lineage that, within our time tree, explained at least in part why leporids radiated and are more species-rich than their sister group.

The diversity trends in Lagomorpha might be related to a competition for resources with rodents. The decline in ochotonid diversity in the Late Miocene coincides with the radiation of the most diverse rodent families, Cricetidae and Muridae, which may have outcompeted the pikas particularly as the strictly herbivorous Arvocolinae murids diversified. Our findings are supported by the fossil evidence that indicate the ochotonid-rodent small body size as ancestral in Glires.

We suggest that Leporidae escaped the competition bottleneck and radiated by evolving a completely new morphospace—large bodies and an efficient cursorial locomotor system—covering wide areas for foraging and outrunning predators more easily than rodents and pikas. Our results indicate that since the Pleistocene, rabbits and hares have radiated, especially those of the genera Lepus and Sylvilagus, which tended to remain taller than most rodents and pikas. Nevertheless, successful leporid body plans have been constrained by larger body sizes, as this would move them into competitive morphospace with ungulate-type herbivores [71].

Conclusions

Multiple markers and fossil priors recovered narrow confidence intervals and a consistently older age for the origin of Lagomorpha around 58 Ma.

Varying the number of fossil calibrations (calibrations sets) had negligible effects on the ages of the major lagomorph lineages.

Our age for the Ochotona ancestor fits the minimum age for the genus in the fossil record, especially because the oldest Ochotona fossil was not included in our calibration set.

Our age for crown Leporidae was estimated at 25.4 Ma with a small confidence interval, thereby reducing the fossil gap to only 12 Myr.

The Leporidae probably scaped competition with Ochotonidae and Muroid rodents when evolved a larger body and a body plan with a cursoriality specialization.

Supporting information

S1 Fig Phylogenetic relationships of Lagomorpha.

Maximum likelihood tree generated using 79 terminals and 5 mitochondrial genes. Nodes with bootstrap values lower than 100 shown. In this topology, all species (including those that were considered rogue taxa) were kept.

(TIF)

S1 Table Species taxonomic assignments based on higher Taxonomy database.

(DOCX)

S2 Table Access codes for sequences for five mitochondrial and 14 nuclear genes used in our analysis.

(PDF)

The authors are thankful for Prof. Lena Geise of the State University of Rio de Janeiro, in Brazil, for a helpful discussion regarding mammalian databases. This study was part of the Master’s thesis of L.I. at the Biodiversity and Biologia Evolutiva Graduate Program at the Federal University of Rio de Janeiro.

10.1371/journal.pone.0307380.r001
Decision Letter 0
Janke Axel Academic Editor
© 2024 Axel Janke
2024
Axel Janke
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
11 Jun 2024

PONE-D-24-20437A molecular time-tree for LagomorphaPLOS ONE

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Additional Editor Comments:

Dear Authors

I am sorry for the delay of the reviewing process, but it is hard to find find suitable and willing reviewer. To speed up the process, I will make my recommendation based on the one reviewer and my expertise.

REVIW OF THE EDITOR

The authors have assembled an impressive amount of sequence data from publicly available resources and have revisited the phylogeny of lagomorphs. This is a timely task, given the rapid accumulation of sequence data.

The analyses are straightforward, robust, and standard, resulting in a classic phylogenetic analysis based on the latest data. Since the focus was on a representative taxon sample, the number of loci (14) is limited, but still sufficient. Time calibration was performed using six fossil calibration points.

The results are nicely put into the context with the concurrent evolution of mammals and plants.

Lagomorpha is a large taxon with a long history of analysis and viewpoints, so my main recommendation/concern for improvement is to better point out differences in current understanding or controversial issues. This would help the non-specialist to better follow your findings.

Perhaps add a conclusion with bullet points for the five (or X) major findings.

Minor Issues:

- Give divergence times consistently with only three digits, because the estimates are not (cannot be) accurate to 10,000 years.

- Clean up the Suppl Fig., e.g. write some support values on the other side of the branch so that the numbers do not overlap.

- Enlarge the major clade to better see the divergence times, e.g. make a separate tree for Lepus. Or remove the outgroup Mus, Rattus, Acomys from the main figure (Fig. 1) to expand the branches for better readability and to show divergences from 30/25 Mya onwards.

These issues lead to a "minor revision" as they only improve the presentation of the results. However, I recommend to follow the recommendations (also of reviewer 1) for a better impact of the nice paper on the field.

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Reviewers' comments:

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Reviewer #1: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: In the presented work, Leandro Iraçabal and coworkers have generated an ultrametric phylogenetic tree for Lagomorpha (hares, rabbits and pikas) using five mitochondrial and 14 nuclear loci. Besides constructing credible molecular phylogeny for the families and genera with high support values for branches, they reveal that the subgenus Ochotona is not monophyletic and would require revision. The tree was calibrated using available fossil evidence, and despite the usual caveats with such timings, seems like a plausible and realistic result.

The introduction is well written and well informed. The analysis used existing sequence data, without additional sampling and sequencing, which explains the targeting few loci in contrast to whole genome analysis. While the approach is perhaps somewhat outdated and does not provide novel sequence information, it is perfectly valid for the purpose and, thanks to the large amounts of available data, the analysis is also very comprehensive. The results are well presented and discussed. The further discussion of the Lagomorph evolution is very nice and highly useful as future reference. I have only a few minor suggestions make:

The title “A molecular time-tree” or the use of “time-tree” in the text appears somewhat imprecise use of terminology. Consider revising to e.g. chronophyletic tree, time-resolved phylogenetic tree or similar.

Introduction:

-Not only the rabbit, but also the European brown hare has been introduced to a number of locations, including Australia.

Results:

It would be highly useful to have more zoomed-in version of the tree in Fig 1 as supplementary figures to better see the estimated speciation times. This is especially interesting in genus Lepus, where the species frequently hybridize. It also better allows to see the effect of the last ice age on the species diversity. These figures could include the dates of the different stages of the Quartenary period.

Discussion:

-Please discuss more the effect of excluding different calibration points and how to evaluate the credibility of the differential results.

-Can the reticulation of especially the Lepus species through frequent hybridization affect the analyses, especially if this also has been occurring in the past?

-It seems likely that the fossil record be biased by the fact that Ochotonidae are much smaller than Leporidae? This could be pointed out.

-The plant codiversification is discussed in detail, but what has been the effect of the last ice age on Lepus radiation? There are a number of cold climate and snow adapted species on both sides of Beringia and the geography of the glacial refugia has probably equally affected the current diversity.

Overall, the manuscript presents a nice contribution towards the understanding of the Lagomorph evolution and origins of its species diversity.

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10.1371/journal.pone.0307380.r002
Author response to Decision Letter 0
Submission Version1
2 Jul 2024

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R: Done. We have included the statement “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript”. Some of the authors received fellowships from funding agencies. This is stated in the Funding Statement section. We could not find the Financial Disclosure section.

5. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Education Ministry of Brazil (CAPES) – Finance Code 001 to student L.I. We are also thankful to the National Research and Technology Council (CNPq) process 310567/2018-1 to C.A.M.R. and a Master’s fellowship to M.R.B.C. and Rio de Janeiro State Research Funding Agency (FAPERJ) processes E-26/010.001887/2019, SEI-260003/001170/2020 (to Michelle Klautau), SEI-260003/012995/2021 to C.A.M.R. and E-26/203.840/2022 (277324) to A.P.S. This study was part of the Master’s thesis of L.I. at the Biodiversity and Biologia Evolutiva Graduate Program at the Federal University of Rio de Janeiro. "We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "The author(s) received no specific funding for this work." Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

R: Done. We have removed the funding statement from the Acknowledgements section.

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R: Done.

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R: Done. Our original crania images were modified from the original copyrighted images, but they have been removed. The remaining images are of our own copyright.

8. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

R: Done

9. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

R: Done.

Additional Editor Comments: Dear Authors. I am sorry for the delay of the reviewing process, but it is hard to find find suitable and willing reviewer. To speed up the process, I will make my recommendation based on the one reviewer and my expertise. REVIEW OF THE EDITOR. The authors have assembled an impressive amount of sequence data from publicly available resources and have revisited the phylogeny of lagomorphs. This is a timely task, given the rapid accumulation of sequence data. The analyses are straightforward, robust, and standard, resulting in a classic phylogenetic analysis based on the latest data. Since the focus was on a representative taxon sample, the number of loci (14) is limited, but still sufficient. Time calibration was performed using six fossil calibration points. The results are nicely put into the context with the concurrent evolution of mammals and plants.

R: Thank you.

Lagomorpha is a large taxon with a long history of analysis and viewpoints, so my main recommendation/concern for improvement is to better point out differences in current understanding or controversial issues. This would help the non-specialist to better follow your findings.

R: Done. To build time-resolved phylogenetic trees from molecular datasets, geological ages are incorporated as calibration priors which are best provided by direct geological source such as the fossil record (primary calibrations). Previous studies used inadequate calibrations such as ages inferred from previous studies (secondary calibrations) and fossils that do not represent the earliest records of extant groups. Such suboptimal practices add much uncertainty to the estimates.

Perhaps add a conclusion with bullet points for the five (or X) major findings.

R: Done. We included bullet point conclusions.

Minor Issues:

- Give divergence times consistently with only three digits, because the estimates are not (cannot be) accurate to 10,000 years.

R: Done. All time estimates are now with one decimal place. (Calibrations were kept with two decimal places.)

- Clean up the Suppl Fig., e.g. write some support values on the other side of the branch so that the numbers do not overlap.

R: Done.

- Enlarge the major clade to better see the divergence times, e.g. make a separate tree for Lepus. Or remove the outgroup Mus, Rattus, Acomys from the main figure (Fig. 1) to expand the branches for better readability and to show divergences from 30/25 Mya onwards.

R: Done. The figure 2 is now a pruned tree for Leporidae.

These issues lead to a "minor revision" as they only improve the presentation of the results. However, I recommend to follow the recommendations (also of reviewer 1) for a better impact of the nice paper on the field.

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:

In the presented work, Leandro Iraçabal and coworkers have generated an ultrametric phylogenetic tree for Lagomorpha (hares, rabbits and pikas) using five mitochondrial and 14 nuclear loci. Besides constructing credible molecular phylogeny for the families and genera with high support values for branches, they reveal that the subgenus Ochotona is not monophyletic and would require revision. The tree was calibrated using available fossil evidence, and despite the usual caveats with such timings, seems like a plausible and realistic result.

R: Thank you.

The introduction is well written and well informed. The analysis used existing sequence data, without additional sampling and sequencing, which explains the targeting few loci in contrast to whole genome analysis. While the approach is perhaps somewhat outdated and does not provide novel sequence information, it is perfectly valid for the purpose and, thanks to the large amounts of available data, the analysis is also very comprehensive. The results are well presented and discussed. The further discussion of the Lagomorph evolution is very nice and highly useful as future reference.

R: Thank you.

I have only a few minor suggestions make: The title “A molecular time-tree” or the use of “time-tree” in the text appears somewhat imprecise use of terminology. Consider revising to e.g. chronophyletic tree, time-resolved phylogenetic tree or similar.

R: Done. We used time-resolved phylogenetic tree.

Introduction:

-Not only the rabbit, but also the European brown hare has been introduced to a number of locations, including Australia.

R: Done. The sentence was rewritten.

Results:

It would be highly useful to have more zoomed-in version of the tree in Fig 1 as supplementary figures to better see the estimated speciation times. This is especially interesting in genus Lepus, where the species frequently hybridize. It also better allows to see the effect of the last ice age on the species diversity. These figures could include the dates of the different stages of the Quartenary period.

R: Done. Figure 2 is the pruned Leporidae from Figure 1.

Discussion:

-Please discuss more the effect of excluding different calibration points and how to evaluate the credibility of the differential results.

R: Done. We have now included a conclusion about the calibration scheme consistency.

-Can the reticulation of especially the Lepus species through frequent hybridization affect the analyses, especially if this also has been occurring in the past?

R: High reticulation levels can certainly affect our analyses, since they were based on a phylogenetic tree which is not an appropriate way to depict the evolution of a reticulated group. However, our discussion and conclusions are probably unaffected as they focused on the macroevolutionary (above genera) patterns in Lagomorpha.

-It seems likely that the fossil record be biased by the fact that Ochotonidae are much smaller than Leporidae? This could be pointed out.

R: Done. Given the same environment, larger animals are more likely to produce fossils simply because their body structures have more volume and therefore more chances to withstand decay until the fossilization process is concluded. However, if that were the case for Lagomorpha, we would expect that the fossil record of leporids would always be richer than the ochotonids’ in sites from multiple continents, that is not the case. Even though the oldest stem leporids are indeed older than the oldest stem ochotonids, the (fossil) diversity of leporids remained considerably low worldwide until the latest Miocene. On the other hand, there is an explosion in ochotonid fossil diversity worldwide from the Oligocene to the late Miocene. Furthermore, the large size of leporids only begin by the Late Miocene, reinforcing that there is no bias in the fossil record favoring this clade because their larger bodies is only a recent event in their

Attachment Submitted filename: Response to reviewer and editor.docx

10.1371/journal.pone.0307380.r003
Decision Letter 1
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Molecular time estimates for the Lagomorpha diversification

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10.1371/journal.pone.0307380.r004
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==== Refs
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