Complete mitochondrial DNA genome sequences of extinct birds: ratite phylogenetics and the vicariance biogeography hypothesis

The ratites have stimulated much debate as to how such large flightless birds came to be distributed across the southern continents, and whether they are a monophyletic group or are composed of unrelated lineages that independently lost the power of flight. Hypotheses regarding the relationships among taxa differ for morphological and molecular data sets, thus hindering attempts to test whether plate tectonic events can explain ratite biogeography. Here, we present the complete mitochondrial DNA genomes of two extinct moas from New Zealand, along with those of five extant ratites (the lesser rhea, the ostrich, the great spotted kiwi, the emu and the southern cassowary and two tinamous from different genera. The non-stationary base composition in these sequences violates the assumptions of most tree-building methods. When this bias is corrected using neighbour-joining with log-determinant distances and non-homogeneous maximum likelihood, the ratites are found to be monophlyletic, with moas basal, as in morphological trees. The avian sequences also violate a molecular clock, so we applied a non-parametric rate smoothing algorithm, which minimizes ancestor-descendant local rate changes, to date nodes in the tree. Using this method, most of the major ratite lineages fit the vicariance biogeography hypothesis, the exceptions being the ostrich and the kiwi, which require dispersal to explain their present distribution.     

Molecular phylogeny and biogeography of Kinosternon flavescens based on complete mitochondrial control region sequences

Nucleotide sequences for the complete mitochondrial control region (1158 bp) were used to determine the phylogenetic relationships among populations of the yellow mud turtle, Kinosternon flavescens (Kinosternidae). Phylogenetic analysis of the mtDNA sequences reveals a polyphyletic K. flavescens with three distinct clades: (1) K. flavescens of the Central Plains, including isolated populations of Illinois and Iowa, (2) K. arizonense in the Sonoran Desert of Arizona and Mexico, and (3) K. durangoense in the Chihuahuan Desert of Durango, Coahuila, and Chihuahua, Mexico. Sequence divergence and nucleotide diversity calculations support a hypothesis of Great Plains K. flavescens dispersal and subsequent isolation of populations in the Midwest related to climatic change during the Pleistocene.     

The molecular systematics and biogeography of the European cobitids based on mitochondrial DNA sequences

Phylogenetic inference regarding the biogeography and evolution of the family Cobitidae depends in large part on the correct interpretation of transitions between the morphological states of secondary sexual characters (e.g., the scale of Canestrini or lamina circularis). Here, we use the complete mitochondrial ATP synthase 8 and 6 and cytochrome b genes to provide an independent assessment of systematics and biogeographic relationships of species in the genus Cobitis, including geographic and subgeneric sampling of species with Canestrini's scale present, duplicated, or absent. The mtDNA-based phylogeny for the genus Cobitis provides the first formal hypothesis for the group and permits a phylogenetic-based assessment of the morphological transitions demonstrated by Canestrini's scale. Our data confirm the monophyly of the genus Cobitis and indicate that European Cobitis comprise six evolutionarily independent lineages. These lineages were defined by nucleotide synapomorphies permitting bootstrapped confidence estimates of 95% or greater and mtDNA genetic distances greater than 4.5% and correspond with moderate fidelity to the Cobitis groups defined by Bacescu (1962, Rev. Roum. Biol. 4, 435-448). The Caucasian lineage, C. cf. sibirica, represents the basal sister species of the genus Cobitis, supporting an eastern Asiatic origin of the European Cobitis: Cobitis sensu stricto, Acanestrinia, Bicanestrinia, Iberocobitis, and Cobitis calderoni. Phylogenetic relationships among Cobitis subgenera and species indicate that the ancestral condition of one scale of Canestrini was duplicated once at the origin of the Bicanestrinia lineage and has been independently lost by C. calderoni and C. elongata. The absence of the scale of Canestrini is the synapomorphy defining the subgenus Acanestrinia, but the mtDNA phylogeny indicates that Acanestrinia is not a natural group and places C. calderoni as the sister lineage to the subgenus Iberocobitis, a finding that is also geographically parsimonius.     

Molecular phylogenetics of Meliaceae (Sapindales) based on nuclear and plastid DNA sequences

Phylogenetic analyses of Meliaceae, including representatives of all four currently recognized subfamilies and all but two tribes (32 genera and 35 species, respectively), were carried out using DNA sequence data from three regions: plastid genes rbcL, matK (partial), and nuclear 26S rDNA (partial). Individual and combined phylogenetic analyses were performed for the rbcL, matK, and 26S rDNA data sets. Although the percentage of informative characters is highest in the segment of matK sequenced, rbcL provides the greatest number of informative characters of the three regions, resulting in the best resolved trees. Results of parsimony analyses support the recognition of only two subfamilies (Melioideae and Swietenioideae), which are sister groups. Melieae are the only tribe recognized previously that are strongly supported as monophyletic. The members of the two small monogeneric subfamilies, Quivisianthe and Capuronianthus, fall within Melioideae and Swietenioideae, respectively, supporting their taxonomic inclusion in these groups. Furthermore, the data indicate a close relationship between Aglaieae and Guareeae and a possible monophyletic origin of Cedreleae of Swietenioideae. For Trichilieae (Melioideae) and Swietenieae (Swietenioideae) lack of monophyly is indicated.     

Molecular phylogenetics of Hypoxidaceae--evidence from plastid DNA data and inferences on morphology and biogeography

Phylogenetic relationships of the monocot family Hypoxidaceae (Asparagales), which occurs mainly in the Southern Hemisphere, were reconstructed using four plastid DNA regions (rbcL, trnL intron, trnL-F intergenic spacer, and trnS-G intergenic spacer) for 56 ingroup taxa including all currently accepted genera and seven species of the closely related families Asteliaceae, Blandfordiaceae, and Lanariaceae. Data were analyzed by applying parsimony, maximum likelihood and Bayesian methods. The intergenic spacer trnS-G--only rarely used in monocot research--contributed a substantial number of potentially parsimony informative characters. Hypoxidaceae consist of three well-supported major clades, but their interrelationships remain unresolved. Our data indicate that in the Pauridia clade one long-distance dispersal event occurred from southern Africa to Australia. Long-distance dispersal scenarios may also be likely for the current distribution of Hypoxis, which occurs on four continents. In the Curculigo clade, the present distribution of Curculigo s.s. on four continents could support a Gondwanan origin, but the level of divergence is too low for this hypothesis to be likely. The main clades correspond well with some floral characters, habit and palynological data, whereas chromosomal data exhibit plasticity and probably result from polyploidization and subsequent dysploidy and/or aneuploidy. Evolutionary flexibility is also suggested by the number of reported pollination syndromes: melittophily, myophily, sapromyophily, and cantharophily. Based on our phylogenetic results, we suggest cautious nomenclatural reorganization to generate monophyly at the generic level.     

Molecular phylogenetics and historical biogeography of Rhinolophus bats

The phylogenetic relationships within the horseshoe bats (genus Rhinolophus) are poorly resolved, particularly at deeper levels within the tree. We present a better-resolved phylogenetic hypothesis for 30 rhinolophid species based on parsimony and Bayesian analyses of the mitochondrial cytochrome b gene and three nuclear introns (TG, THY and PRKC1). Strong support was found for the existence of two geographic clades within the monophyletic Rhinolophidae: an African group and an Oriental assemblage. The relaxed Bayesian clock method indicated that the two rhinolophid clades diverged approximately 35 million years ago and results from Dispersal Vicariance (DIVA) analysis suggest that the horseshoe bats arose in Asia and subsequently dispersed into Europe and Africa.     

Colonization history of Scots pine in Eastern Europe and North Asia based on mitochondrial DNA variation

During Quaternary glaciations, the ranges of Northern Eurasia forest species periodically experienced contraction followed by subsequent re-colonizations in the interglacial intervals. However, unlike the broadleaf trees of temperate forests, taiga species seem not to have retreated fully to southern regions in unfavorable periods and possibly survived at mid-latitudes in multiple refugia. Here, we report a study of genetic variation of three mitochondrial DNA markers in 90 populations of Scots pine (Pinus sylvestris) located from Eastern Europe to Eastern Siberia. The geographic distribution of seven mitotypes demonstrated the split between western and eastern populations approximately along the 38th meridian. Genetic diversity in the western part was significantly higher than in the eastern one. Five mitotypes were western- and one eastern-specific. One mitotype was common in both regions, but in the eastern part it occurred only in the South Urals and adjacent areas. The geographic structure in the mitotype distribution supports a hypothesis of post-glacial re-colonization of the studied territory from the European and Ural refugia.     

Molecular biogeography of red deer Cervus elaphus from eastern Europe: insights from mitochondrial DNA sequences

European red deer are known to show a conspicuous phylogeographic pattern with three distinct mtDNA lineages (western, eastern and North-African/Sardinian). The western lineage, believed to be indicative of a southwestern glacial refuge in Iberia and southern France, nowadays covers large areas of the continent including the British Isles, Scandinavia and parts of central Europe, while the eastern lineage is primarily found in southeast-central Europe, the Carpathians and the Balkans. However, large parts of central Europe and the whole northeast of the continent were not covered by previous analyses. To close this gap, we produced mtDNA control region sequences from more than 500 red deer from Denmark, Germany, Poland, Lithuania, Belarus, Ukraine and western Russia and combined our data with sequences available from earlier studies to an overall sample size of almost 1,100. Our results show that the western lineage extends far into the European east and is prominent in all eastern countries except for the Polish Carpathians, Ukraine and Russia where only eastern haplotypes occurred. While the latter may actually reflect the natural northward expansion of the eastern lineage after the last ice age, the present distribution of the western lineage in eastern Europe may in large parts be artificial and a result of translocations and reintroduction of red deer into areas where the species became extinct in historical times.     

Molecular phylogenetics and biogeography of Neotropical tanagers in the genus Tangara

Species in the genus Tangara are distributed throughout the New World tropics and vary in their morphology, behavior, and ecology. We used data from the cytochrome b and ND 2 genes to provide the first phylogenetic perspective on the evolution of this diversity. Reconstructions based on parsimony, maximum likelihood, and Bayesian approaches were largely congruent. The genus is monophyletic and consists of two main clades. Within these clades, DNA sequence data confirm the monophyly of most previously recognized species groups within Tangara, indicating general concordance between molecular data and impressions based on geographic distribution, morphology, and behavior. Within some currently recognized species, levels of DNA sequence variation are larger than expected, suggesting multiple taxa may be involved. In contrast, some currently recognized species are only weakly differentiated from their sister species. Biogeographic analyses indicate that many early speciation events occurred in the Andes. More recently, dispersal events followed by subsequent speciation have occurred in other geographic areas of the Neotropics. Assuming a molecular clock, most speciation events occurred well before Pleistocene climatic cycles. The time frame of Tangara speciation corresponds more closely to a period of continued uplift in the Andes during the late Miocene and Pliocene.     

Molecular phylogenetics of the bee-eaters (Aves: Meropidae) based on nuclear and mitochondrial DNA sequence data

The bee-eaters (family Meropidae) comprise a group of brightly colored, but morphologically homogeneous, birds with a wide variety of life history characteristics. A phylogeny of bee-eaters was reconstructed using nuclear and mitochondrial DNA sequence data from 23 of the 25 named bee-eater species. Analysis of the combined data set provided a well-supported phylogenetic hypothesis for the family. Nyctiornis is the sister taxon to all other bee-eaters. Within the genus Merops, we recovered two well-supported clades that can be broadly separated into two groups along geographic and ecological lines, one clade with mostly African resident species and the other clade containing a mixture of African and Asian taxa that are mostly migratory species. The clade containing resident African species can be further split into two groups along ecological lines by habitat preference into lowland forest specialists and montane forest and forest edge species. Intraspecific sampling in several of the taxa revealed moderate to high (3.7-6.5%, ND2) levels of divergence in the resident taxa, whereas the lone migratory taxon showed negligible levels of intraspecific divergence. This robust molecular phylogeny provides the phylogenetic framework for future comparative tests of hypotheses about the evolution of plumage patterns, sociality, migration, and delayed breeding strategies.     

Molecular genetic differentiation of ethnic populations in Southern and Eastern Siberia based on mitochondrial DNA polymorphism

Using the data on mitochondrial DNA (mtDNA) polymorphism, genetic structures of the ethnic groups inhabiting South and East Siberia, including Altaians, Buryats, Tuvinians, Todjins, Tofalars, Yakuts, and Evenks were described. Mitochondrial gene pools of the populations examined were characterized by different ratios between Mongoloid (M*, C, D, E/G, G, A, B, and F) and Caucasoid (H, HV, I, J, K, T, U, and X) mtDNA lineages. All the populations studied carried a marked Mongoloid component, maximum frequency of which was observed in Evenks (92.4%) and Buryats (90.1%). Maximum frequencies of Caucasoid mtDNA lineages were detected in Tofalars (20.7%) and Yakuts (14.5%). Statistically significant interpopulation differences regarding the frequencies of mtDNA haplogroups were observed between all populations examined, excluding the pairs of Evenks-Yakuts, Evenks-Tuvinians, and Tuvinians-Todjins. Differentiation of the ethnic groups inhabiting South and East Siberia, as well as Central and Middle Asia, is discussed based on genetic, linguistic, and anthropological data.     

Molecular phylogenetics of the Peromyscus boylii species group (Rodentia: muridae) based on mitochondrial cytochrome b sequences

Variation in the mitochondrial cytochrome b gene (1143 bp) was examined to estimate the phylogenetic relationships of taxa within the Peromyscus boylii species group. In addition, phylogenetic relationships among the aztecus, boylii, and truei species groups were addressed. Maximum-likelihood, neighbor-joining, and maximum-parsimony (weighted and equally weighted) analyses produced similar topologies with P. boylii, P. beatae, P. simulus, P. stephani, P. madrensis, P. levipes, and three undescribed taxa from western Mexico forming a monophyletic unit. At least two of the undescribed taxa from western Mexico potentially represent species. Members of the P. aztecus species group formed a clade separate from the P. boylii group and should be recognized as a distinct species group. P. sagax, P. polius, and P. pectoralis, formerly placed in the P. boylii species group, generally formed an unresolved polytomy with the P. truei, P. aztecus, and P. boylii species groups. P. attwateri formed a sister taxon relationship with members of the P. truei species group (P. difficilis and P. nasutus) and should be considered a member of this group. Members of the P. truei species group did not form a monophyletic unit, indicating that this species group is not monophyletic and may be composed of two assemblages.     

Molecular phylogenetics of Ruscaceae sensu lato and related families (Asparagales) based on plastid and nuclear DNA sequences

Background

Previous phylogenetics studies of Asparagales, although extensive and generally well supported, have left several sets of taxa unclearly placed and have not addressed all relationships within certain clades thoroughly (some clades were relatively sparsely sampled). One of the most important of these is sampling within and placement of Nolinoideae (Ruscaceae s.l.) of Asparagaceae sensu Angiosperm Phylogeny Group (APG) III, which subfamily includes taxa previously referred to Convallariaceae, Dracaenaaceae, Eriospermaceae, Nolinaceae and Ruscaceae.

Methods

A phylogenetic analysis of a combined data set for 126 taxa of Ruscaceae s.l. and related groups in Asparagales based on three nuclear and plastid DNA coding genes, 18S rDNA (1796 bp), rbcL (1338 bp) and matK (1668 bp), representing a total of approx. 4·8 kb is presented. Parsimony and Bayesian inference analyses were conducted to elucidate relationships of Ruscaceae s.l. and related groups, and parsimony bootstrap analysis was performed to assess support of clades.

Key Results

The combination of the three genes results in the most highly resolved and strongly supported topology yet obtained for Asparagales including Ruscaceae s.l. Asparagales relationships are nearly congruent with previous combined gene analyses, which were reflected in the APG III classification. Parsimony and Bayesian analyses yield identical relationships except for some slight variation among the core asparagoid families, which nevertheless form a strongly supported group in both types of analyses. In core asparagoids, five major clades are identified: (1) Alliaceae s.l. (sensu APG III, Amarylidaceae–Agapanthaceae–Alliaceae); (2) Asparagaceae–Laxmanniaceae–Ruscaceae s.l.; (3) Themidaceae; (4) Hyacinthaceae; (5) Anemarrhenaceae–Behniaceae–Herreriaceae–Agavaceae (clades 2–5 collectively Asparagaceae s.l. sensu APG III). The position of Aphyllanthes is labile, but it is sister to Themidaceae in the combined maximum-parsimony tree and sister to Anemarrhenaceae in the Bayesian analysis. The highly supported clade of Xanthorrhoeaceae s.l. (sensu APG III, including Asphodelaceae and Hemerocallidaceae) is sister to the core asparagoids. Ruscaceae s.l. are a well-supported group. Asparagaceae s.s. are sister to Ruscaceae s.l., even though the clade of the two families is weakly supported; Laxmanniaceae are strongly supported as sister to Ruscaceae s.l. and Asparagaceae. Ruscaceae s.l. include six principal clades that often reflect previously named groups: (1) tribe Polygonateae (excluding Disporopsis); (2) tribe Ophiopogoneae; (3) tribe Convallarieae (excluding Theropogon); (4) Ruscaceae s.s. + Dracaenaceae + Theropogon + Disporopsis + Comospermum; (5) Nolinaceae, (6) Eriospermum.

Conclusions

The analyses here were largely conducted with new data collected for the same loci as in previous studies, but in this case from different species/DNA accessions and greater sampling in many cases than in previously published analyses; nonetheless, the results largely mirror those of previously conducted studies. This demonstrates the robustness of these results and answers questions often raised about reproducibility of DNA results, given the often sparse sampling of taxa in some studies, particularly the earliest ones. The results also provide a clear set of patterns on which to base a new classification of the subfamilies of Asparagaceae s.l., particularly Ruscaceae s.l. (= Nolinoideae of Asparagaceae s.l.), and examine other putatively important characters of Asparagales.     

Phylogenetic relationships and biogeography of the genus Chondrostoma inferred from mitochondrial DNA sequences

A phylogeny of the species of the nase genus Chondrostoma was constructed from a complete mitochondrial cytochrome b gene (1140 bp). Molecular phylogeny was used to revise the current systematics of this group, and to infer a biogeographical model of the Mediterranean area during the Cenozoic period. We confirmed the monophyly of the genus Chondrostoma, and defined seven different lineages within it: Polylepis, Arcasii, Lemmingii, Toxostoma, Nasus, C. genei, and C. soetta. The separation of main lineages within Chondrostoma occurred in the Middle-Upper Miocene, approximately 11 million years ago, while the greatest species radiation took place in the Pliocene close to the time the current drainages system were created. It is unlikely that this genus experienced an extensive dispersal during the Messinian, in the Lago-Mare Phase. Given the level of current knowledge, a biogeographical model constructed on the basis of vicariant events seems more realistic than does a dispersalist model.     

Molecular phylogenetics and historical biogeography of Hawaiian Dryopteris (Dryopteridaceae)

The fern genus Dryopteris (Dryopteridaceae) is represented in the Hawaiian Islands by 18 endemic taxa and one non-endemic, native species. The goals of this study were to determine whether Dryopteris in Hawai'i is monophyletic and to infer the biogeographical origins of Hawaiian Dryopteris by determining the geographical distributions of their closest living relatives. We sequenced two chloroplast DNA fragments, rbcL and the trnL-F intergenic spacer (IGS), for 18 Hawaiian taxa, 45 non-Hawaiian taxa, and two outgroup species. For individual fragments, we estimated phylogenetic relationships using Bayesian inference and maximum parsimony. We performed a combined analysis of both cpDNA fragments employing Bayesian inference, maximum parsimony, and maximum likelihood. These analyses indicate that Hawaiian Dryopteris is not monophyletic, and that there were at least five separate colonizations of the Hawaiian Islands by different species of dryopteroid ferns, with most of the five groups having closest relatives in SE Asia. The results suggest that one colonizing ancestor, perhaps from SE Asia, gave rise to eight endemic taxa (the glabra group). Another colonizing ancestor, also possibly from SE Asia, gave rise to a group of five endemic taxa (the exindusiate group). Dryopteris fusco-atra and its two varieties, which are endemic to Hawai'i, most likely diversified from a SE Asian ancestor. The Hawaiian endemic Nothoperanema rubiginosum has its closest relatives in SE Asia, and while the remaining two species, D. wallichiana and D. subbipinnata, are sister species, their biogeographical origins could not be determined from these analyses due to the widespread distributions of D. wallichiana and its closest non-Hawaiian relative.     

Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences

To study the inter- and infrafamilial phylogenetic relationships in the order Caryophyllales sensu lato (s.l.), ～930 base pairs of the matK plastid gene have been sequenced and analyzed for 127 taxa. In addition, these sequences have been combined with the rbcL plastid gene for 53 taxa and with the rbcL and atpB plastid genes as well as the nuclear 18S rDNA for 26 taxa to provide increased support for deeper branches. The red pigments of Corbichonia, Lophiocarpus, and Sarcobatus have been tested and shown to belong to the betacyanin class of compounds. Most taxa of the order are clearly grouped into two main clades (i.e., "core" and "noncore" Caryophyllales) which are, in turn, divided into well-defined subunits. Phytolaccaceae and Molluginaceae are polyphyletic, and Portulacaceae are paraphyletic, whereas Agdestidaceae, Barbeuiaceae, Petiveriaceae, and Sarcobataceae should be given familial recognition. Two additional lineages are potentially appropriate to be elevated to the family level in the future: the genera Lophiocarpus and Corbichonia form a well-supported clade on the basis of molecular and chemical evidence, and Limeum appears to be separated from other Molluginaceae based on both molecular and ultrastructural data.     

Phylogenetic relationships and biogeography of xantusiid lizards, inferred from mitochondrial DNA sequences.

Portions of two mitochondrial genes (12S ribosomal RNA and cytochrome b) were sequenced in seven species to examine phylogenetic relationships within the lizard family Xantusiidae. Phylogenies derived from these sequences (709 total bp) are concordant and indicate that the Cuban species Cricosaura typica is the sister group to all other xantusiids. The Middle American genus Lepidophyma is the closest relative of Xantusia, and X. riversiana (California Islands) the closest relative of X. vigilis (mainland). These findings are not in agreement either with the results of a recent morphological analysis that united Cricosaura and Lepidophyma as closest relatives or with past studies that have recognized X. riversiana as a separate genus. Levels of sequence divergence, as well as the age and affinities of some mainland fossil taxa, suggest that the origin of Cricosaura was associated with the tectonic evolution of the Greater Antilles in the late Cretaceous. These results further demonstrate that significant resolution of phylogenies can be obtained with relatively short DNA sequences and that these mitochondrial genes are concordant in their estimation of phylogeny.     

Molecular phylogenetics of the sexually deceptive orchid genus Ophrys (Orchidaceae) based on nuclear and chloroplast DNA sequences.

We present a phylogenetic analysis of the major lineages of the sexually deceptive orchid genus Ophrys based on nuclear ribosomal (nr) DNA (internal transcribed spacer region) and noncoding chloroplast (cp) DNA (trnL-trnF region) sequences. Sequence divergence within and among major Ophrys lineages was low for both nrDNA and cpDNA sequences. Separate analyses resulted in similar but poorly resolved trees. An incongruence length difference test revealed that nrDNA and cpDNA data sets were not incongruent. A combined analysis resulted in a better-resolved phylogenetic hypothesis of relationships among the major Ophrys lineages. Our data strongly support a division of Ophrys into two groups. These groups do not correspond to the earlier proposed sections Euophrys and Pseudophrys and are thus in conflict with traditional classifications. Our results support a well-resolved monophyletic group that contains the geographically widespread O. bombyliflora, O. speculum, O. tenthredinifera, and the O. fusca-lutea lineage. Relationships in the other group are poorly resolved. Based on our observations that taxa with identical sequences at presumably rapidly evolving loci clearly differ in floral morphology, we hypothesize that the diversity in the genus Ophrys is the result of a recent radiation in this orchid lineage.     

Molecular phylogeny of the lemur family cheirogaleidae (primates) based on mitochondrial DNA sequences

Cheirogaleidae currently comprises five genera whose relationships remain contentious. The taxonomic status and phylogenetic position of both Mirza coquereli and Allocebus trichotis are still unclear. The taxonomic status of the recently discovered Microcebus ravelobensis (a sympatric sibling species of Microcebus murinus) and its phylogenetic position also require further examination. A approximately 2.4-kb mitochondrial DNA sequence including part of the COIII gene, complete ND3, ND4L, and ND4 genes, and 5 tRNAs was used to clarify relationships among cheirogaleids. Mirza and Microcebus form a clade representing the sister group of Allocebus, with a clade containing Cheirogaleus major and Cheirogaleus medius diverging first. M. ravelobensis and Microcebus rufus form a subclade within Microcebus, with M. murinus as its sister group. The molecular data support the generic status of Mirza coquereli and species-level divergence of M. ravelobensis. Furthermore, "M. rufus" may well represent more than one species.