Molecular support for Afrotheria and the polyphyly of Lipotyphla based on analyses of the growth hormone receptor gene

The order Lipotyphla has generally been viewed as a difficult group to classify. For example, recent morphologically based analyses only weakly support the lipotyphla while molecular evidence renders it polyphyletic, placing the golden moles and tenrecs in the superorder known as Afrotheria. Afrotheria is an hypothesized order that contains elephants, sirenians, hyraxes, aardvarks, elephant shrews, tenrecs, and golden moles. Within this group, it has been suggested that the African lipotyphlans (tenrecs and golden moles) form a monophyletic order sometimes referred to as "Afroscoricida," but more appropriately termed Tenrecoidea. The paper presents a molecular analysis of 36 taxa including representatives of five of the six families in Lipotyphla (Solenodontidae is absent) and all orders within Afrotheria. Parsimony analyses were completed using data from the nucleotide sequence of the tenth exon of the growth hormone receptor gene (GHR). These analyses support both the polyphyly of Lipotyphla and the monophyly of Afrotheria with high bootstrap and jackknife support. In addition, the remaining lipotyphlans (known as Eulipotyphla) appear polyphyletic, as does Tenrecoidea.     

Convergence vs. Specialization in the ear region of moles (mammalia)

We investigated if and how the inner ear region undergoes similar adaptations in small, fossorial, insectivoran‐grade mammals, and found a variety of inner ear phenotypes. In our sample, afrotherian moles (Chrysochloridae) and the marsupial Notoryctes differ from most other burrowing mammals in their relatively short radii of semicircular canal curvature; chrysochlorids and fossorial talpids share a relatively long interampullar width. Chrysochlorids are unique in showing a highly coiled cochlea with nearly four turns. Extensive cochlear coiling may reflect their greater ecological dependence on low frequency auditory cues compared to talpids, tenrecids, and the marsupial Notoryctes. Correspondingly, the lack of such extensive coiling in the inner ear of other fossorial species may indicate a greater reliance on other senses to enable their fossorial lifestyle, such as tactile sensation from vibrissae and Eimer's organs. The reliance of chrysochlorids on sound is evident in the high degree of coiling and in the diversity of its mallear types, and may help explain the lack of any semiaquatic members of that group. The simplest mallear types among chrysochlorids are not present in the basal‐most members of that clade, but all extant chrysochlorids investigated to date exhibit extensive cochlear coiling. The chrysochlorid ear region thus exhibits mosaic evolution; our data suggest that extensive coiling evolved in chrysochlorids prior to and independently of diversification in middle ear ossicle size and shape. J. Morphol. 276:900–914, 2015. © 2015 Wiley Periodicals, Inc.     

Relationships of Endemic African Mammals and Their Fossil Relatives Based on Morphological and Molecular Evidence

Analyses of anatomical and DNA sequence data run on a parallel supercomputer that include fossil taxa support the inclusion of tenrecs and golden moles in the Afrotheria, an endemic African clade of placental mammals. According to weighting schemes of morphological and molecular data that maximize congruence, extinct members of the afrotherian crown group include embrithopods, Plesiorycteropus, desmostylians, and the condylarths Hyopsodus, Meniscotherium, and possibly Phenacodus. By influencing the optimization of anatomical characters, molecular data have a large influence on the relationships of several extinct taxa. The inclusion of fossils and morphological data increases support for an elephant-sea cow clade within Paenungulata and identifies ancient, northern elements of a clade whose living members in contrast suggest an historically Gondwanan distribution. In addition, maximally congruent topologies support the position of Afrotheria as well-nested, not basal, within Placentalia. This pattern does not accord with the recent hypothesis that the divergence of placental mammals co-occurred with the tectonic separation of Africa and South America.     

Early Tertiary mammals from North Africa reinforce the molecular Afrotheria clade

The phylogenetic pattern and timing of the radiation of mammals, especially the geographical origins of major crown clades, are areas of controversy among molecular biologists, morphologists and palaeontologists. Molecular phylogeneticists have identified an Afrotheria clade, which includes several taxa as different as tenrecs (Tenrecidae), golden moles (Chrysochloridae), elephant-shrews (Macroscelididae), aardvarks (Tubulidentata) and paenungulates (elephants, sea cows and hyracoids). Molecular data also suggest a Cretaceous African origin for Afrotheria within Placentalia followed by a long period of endemic evolution on the Afro-Arabian continent after the mid-Cretaceous Gondwanan breakup (approx. 105-25 Myr ago). However, there was no morphological support for such a natural grouping so far. Here, we report new dental and postcranial evidence of Eocene stem hyrax and macroscelidid from North Africa that, for the first time, provides a congruent phylogenetic view with the molecular Afrotheria clade. These new fossils imply, however, substantial changes regarding the historical biogeography of afrotheres. Their long period of isolation in Africa, as assumed by molecular inferences, is now to be reconsidered inasmuch as Eocene paenungulates and elephant-shrews are here found to be related to some Early Tertiary Euramerican 'hyopsodontid condylarths' (archaic hoofed mammals). As a result, stem members of afrotherian clades are not strictly African but also include some Early Paleogene Holarctic mammals.     

Congruence between comparative morphology and molecular phylogenies: Evolution of the male genital skeletal/muscular system in the subtribe Polyommatina (Lepidoptera,Lycaenidae)

The skeleton and musculature of male genitalia were studied in species of a model butterfly group (subtribe Polyommatina, Lycaenidae). In total, we analyzed 45 species of the tribe Polyommatini most of which were previously used in the molecular phylogenetic study (Talavera et al., 2013). The studied morphological characters were mapped on the molecular trees, which allowed us to reveal trends of morphological changes and to estimate the age of their origin. As a result, chronology of evolution of skeleton and musculature traits was established. It was shown that periods of slow morphological evolution alternated in the subtribe Polyommatina with those of a high rate of origin of new traits. For example, topography of the intravalvar muscles has not changed for 26 MY preserving their initial fan-shaped attachment. The evolution of intravalvar muscles started 10 MYA, proceeded slowly during the first 5 MY, and then accelerated during the last 5 MY resulting in the extensive splitting of the musculature in most monophyletic lineages. Mapping the morphological characters on the phylogeny demonstrated that the rates of skeleton and muscle evolution within the skeleton/musculature apparatus were different. In most cases the intravalvar musculature evolved much faster than the skeleton. The cladistic interpretation of states of morphological traits was found to be consistent with phylogenetic reconstructions based on analysis of multiple molecular markers. Moreover, morphological synapomorphies were found for the lineages Alpherakya + Glabroculus and Aricia + (Alpherakya + Glabroculus), which had low statistical support in molecular phylogenetic analysis. Additionally, in some cases molecular studies helped to reveal trends in the evolution of morphological traits. For example, the unpaired uncus and the compact juxta are not plesiomorphic for Cupidina as previously thought; instead, they were shown to have evolved secondarily within this subtribe.     

"Lipotyphlan" phylogeny based on the growth hormone receptor gene: a reanalysis

From an evolutionary perspective, "insectivores" have been one of the most important mammalian groups for over a century. Morphologists have successively pruned flying lemurs, elephant shrews, and tree shrews from Insectivora, but have retained chrysochlorids, tenrecs, erinaceids, soricids, talpids, and solenodontids in crown-group Lipotyphla. With the appearance of large molecular data sets, the monophyly of Lipotyphla has proved untenable. Rather, an emerging consensus is that Lipotyphla is a diphyletic taxon comprised of two monophyletic groups, Afrosoricida and Eulipotyphla. A recent paper by Malia et al. [Mol. Phylogenet. Evol. 24 (2002) 91-101] challenged this view and argued that "While the data [Growth Hormone Receptor] were unable to support the orders Lipotyphla, Eulipotyphla, and Tenrecoidea [= Afrosoricida] this was most likely due to the polyphyly of these groups and not to problems associated with the gene itself such as saturation or highly divergent sequences em leader " (p. 100). We analyzed Malia et al.'s original GHR data set (at both nuclear and protein level), an expanded GHR data set that included 49 additional sequences, and a concatenated data set that included GHR, BRCA1, vWF, and A2AB for a diverse selection of lipotyphlan taxa. Although protein analyses proved inconclusive, all analyses at the DNA level clearly show that the statement of Malia et al. is erroneous. Indeed, likelihood analyses with GHR and with the concatenated data set provide more support for Eulipotyphla and Afrosoricida than for competing hypotheses. These results also highlight the potential pitfalls of single-gene and parsimony-only analyses.     

Afrotherian phylogeny as inferred from complete mitochondrial genomes

Afrotheria is a huge assemblage of various mammals encompassing six orders that were once classified as distantly related groups. This superordinal relationship may have resulted from the break-up of Gondowanaland followed by the isolation of the African continent between 105 and 40 million years ago. Although the monophyly of Afrotheria is well supported by recent molecular studies, the interrelationships within afrotherian mammals remain unclarified. In this study, we determined the sequence of the complete mitochondrial genomes of hyrax, golden mole, and elephant shrew. These sequences were compared with those of other eutherians to analyze the phylogenetic relationships among afrotherians and, in particular, those among paenungulates. Our mitochondrial genome analysis supports the monophyly of Tethytheria.     

A new estimate of afrotherian phylogeny based on simultaneous analysis of genomic,morphological, and fossil evidence

Background  

The placental mammalian clade Afrotheria is now supported by diverse forms of genomic data, but interordinal relationships within, and morphological support for, the group remains elusive. As a means for addressing these outstanding problems, competing hypotheses of afrotherian interordinal relationships were tested through simultaneous parsimony analysis of a large data set (> 4,590 parsimony informative characters) containing genomic data (> 17 kb of nucleotide data, chromosomal associations, and retroposons) and 400 morphological characters scored across 16 extant and 35 extinct afrotherians.     

Chromosomal instability in Afrotheria: fragile sites,evolutionary breakpoints and phylogenetic inference from genome sequence assemblies

Background  

Extant placental mammals are divided into four major clades (Laurasiatheria, Supraprimates, Xenarthra and Afrotheria). Given that Afrotheria is generally thought to root the eutherian tree in phylogenetic analysis of large nuclear gene data sets, the study of the organization of the genomes of afrotherian species provides new insights into the dynamics of mammalian chromosomal evolution. Here we test if there are chromosomal bands with a high tendency to break and reorganize in Afrotheria, and by analyzing the expression of aphidicolin-induced common fragile sites in three afrotherian species, whether these are coincidental with recognized evolutionary breakpoints.     

Phylogeny and conservation priorities of afrotherian mammals (Afrotheria,Mammalia)

Kuntner, M., May‐Collado, L. J. & Agnarsson, I. (2010). Phylogeny and conservation priorities of afrotherian mammals (Afrotheria, Mammalia). —Zoologica Scripta, 40, 1–15. Phylogenies play an increasingly important role in conservation biology providing a species‐specific measure of biodiversity – evolutionary distinctiveness (ED) or phylogenetic diversity (PD) – that can help prioritize conservation effort. Currently, there are many available methods to integrate phylogeny and extinction risk, with an ongoing debate on which may be best. However, the main constraint on employing any of these methods to establish conservation priorities is the lack of detailed species‐level phylogenies. Afrotheria is a recently recognized clade grouping anatomically and biologically diverse placental mammals: elephants and mammoths, dugong and manatees, hyraxes, tenrecs, golden moles, elephant shrews and aardvark. To date, phylogenetic studies have focused on understanding higher level relationships among the major groups within Afrotheria. Here, we provide a species‐level phylogeny of Afrotheria based on nine molecular loci, placing nearly 70% of the extant afrotherian species (50) and five extinct species. We then use this phylogeny to assess conservation priorities focusing on the widely used evolutionary distinctiveness and global endangeredness (EDGE) method and how that compares to the more recently developed PD framework. Our results support the monophyly of Afrotheria and its sister relationship to Xenarthra. Within Afrotheria, the basal division into Afroinsectiphilia (aardvark, tenrecs, golden moles and elephant shrews) and Paenungulata (hyraxes, dugongs, manatees and elephants) is supported, as is the monophyly of all afrotherian families: Elephantidae, Procaviidae, Macroscelididae, Chrysochloridae, Tenrecidae, Trichechidae and Dugongidae. Within Afroinsectiphilia, we recover the most commonly proposed topology (Tubulidentata sister to Afroscoricida plus Macroscelidea). Within Paenungulata, Sirenia is sister to Hyracoidea plus Proboscidea, a controversial relationship supported by morphology. Within Proboscidea, the mastodon is sister to the remaining elephants and the woolly mammoth sister to the Asian elephant, while both living elephant genera, Loxodonta and Elephas are paraphyletic. Top ranking evolutionarily unique species always included the aardvark, followed by several species of elephant shrews and tenrecs. For conservation priorities top ranking species always included the semi‐aquatic Nimba otter shrew, some poorly known species, such as the Northern shrew tenrec, web‐footed tenrec, giant otter shrew and Giant golden mole, as well as high profile conservation icons like Asian elephant, dugong and the three species of manatee. Conservation priority analyses were broadly congruent between the EDGE and PD methodologies. However, for certain species EDGE overestimates conservation urgency as it, unlike PD, fails to account for the status of closely related, but less threatened, species. Therefore, PD offers a better guide to conservation decisions.     

Molecular estimation of eulipotyphlan divergence times and the evolution of "Insectivora"

"Insectivores" are one of the key groups in understanding mammalian origins. For years, systematics of "Lipotyphla" taxa remained extremely unstable and challenged. Today, with the application of molecular techniques, "Lipotyphla" appears to be a paraphyletic assemblage that encompasses hedgehogs, shrews, and moles (i.e., Eulipotyphla-a member of Laurasiatheria), and golden moles and tenrecs (i.e., Afrosoricida-a member of Afrotheria). Based on nuclear genes and on this well-established phylogenetic framework, we estimated Bayesian relaxed molecular clock divergence times among major lineages of "Lipotyphla." Crown placental mammals are shown to diversify 102+/-6 million years ago (Mya; mean+/-one standard-deviation), followed by Boreoeutheria (94+/-6 Mya), Laurasiatheria (85+/-5 Mya), and Eulipotyphla (73+/-5), with moles separating from hedgehogs+shrews just at the K/T boundary (65+/-5 Mya). During the Early and Middle Eocene, all extant eulipotyphlan subfamilies originated: Uropsilinae (52+/-5 Mya), and Desmaninae, Talpinae, Erinaceinae, Hylomyinae, Soricinae, and Crocidurinae (38-42+/-5 Mya). Afrosoricida separated from Macroscelidae 69+/-5 Mya, golden moles from tenrecs 63+/-5 Mya, and the diversification within tenrecs occurred 43+/-5 Mya. Divergence times are shown to be in reasonably good agreement with the fossil record of eulipotyphlans, but not with the one of afrosoricid "insectivores." Eulipotyphlans diversification might have been sculpted by variations in paleoclimates of the cenozoic era.     

Genome Size: A Novel Genomic Signature in Support of Afrotheria

Molecular phylogenetic analyses suggest an emerging phylogeny for the extant Placentalia (eutherian) that radically departs from morphologically based constructions of the past. Placental mammals are partitioned into four supraordinal clades: Afrotheria, Xenarthra, Laurasiatheria, and Euarchontoglires. Afrotheria form an endemic African clade that includes elephant shrews, golden moles, tenrecs, aardvarks, hyraxes, elephants, dugongs, and manatees. Datamining databases of genome size (GS) shows that till today just one afrotherian GS has been evaluated, that of the aardvark Orycteropus afer. We show that the GSs of six selected representatives across the Afrotheria supraordinal group are among the highest for the extant Placentalia, providing a novel genomic signature of this enigmatic group. The mean GS value of Afrotheria, 5.3 ± 0.7 pg, is the highest reported for the extant Placentalia. This should assist in planning new genome sequencing initiatives. [Reviewing Editor: Dmitri Petrov]     

Tenrec phylogeny and the noninvasive extraction of nuclear DNA

Due in part to scarcity of material, no published study has yet cladistically addressed the systematics of living and fossil Tenrecidae (Mammalia, Afrotheria). Using a noninvasive technique for sampling nuclear DNA from museum specimens, we investigate the evolution of the Tenrecidae and assess the extent to which tenrecids fit patterns of relationships proposed for other terrestrial mammals on Madagascar. Application of several tree-reconstruction techniques on sequences of the nuclear growth hormone receptor gene and morphological data for all recognized tenrecid genera supports monophyly of Malagasy tenrecids to the exclusion of the two living African genera. However, both parsimony and Bayesian methods favor a close relationship between fossil African tenrecs and the Malagasy Geogale, supporting the hypothesis of island paraphyly, but not polyphyly. More generally, the noninvasive extraction technique can be applied with minimal risk to rare/unique specimens and, by better utilizing museum collections for genetic work, can greatly mitigate field expenses and disturbance of natural populations.     

Dental eruption in afrotherian mammals

Background  

Afrotheria comprises a newly recognized clade of mammals with strong molecular evidence for its monophyly. In contrast, morphological data uniting its diverse constituents, including elephants, sea cows, hyraxes, aardvarks, sengis, tenrecs and golden moles, have been difficult to identify. Here, we suggest relatively late eruption of the permanent dentition as a shared characteristic of afrotherian mammals. This characteristic and other features (such as vertebral anomalies and testicondy) recall the phenotype of a human genetic pathology (cleidocranial dysplasia), correlations with which have not been explored previously in the context of character evolution within the recently established phylogeny of living mammalian clades.     

Molecular phylogenetic evidence confirming the Eulipotyphla concept and in support of hedgehogs as the sister group to shrews

For more than a century, living insectivore-like mammals have been viewed as little removed from the ancestral mammalian stock based on their retention of numerous primitive characteristics. This circumstance has made "insectivores" a group of special interest in the study of mammalian evolution. included hedgehogs, moles, shrews, solenodons, golden moles, tenrecs, flying lemurs, tree shrews, and elephant shrews in Insectivora. Subsequently, morphologists excluded flying lemurs, tree shrews, and elephant shrews from Insectivora and placed these taxa in the orders Dermoptera, Scandentia, and Macroscelidea, respectively. The remaining insectivores constitute Lipotyphla, which is monophyletic based on morphology. In contrast, molecular data suggest that lipotyphlans are polyphyletic, with golden moles and tenrecs placed in their own order (Afrosoricida) in the superordinal group Afrotheria. Studies based on nuclear genes support the monophyly of the remaining lipotyphlans (=Eulipotyphla) whereas mitochondrial genome studies dissociate hedgehogs from moles and place the former as the first offshoot on the placental tree. One shortcoming of previous molecular studies investigating lipotyphlan relationships is limited taxonomic sampling. Here, we evaluate lipotyphlan relationships using the largest and taxonomically most diverse data set yet assembled for Lipotyphla. Our results provide convincing support for both lipotyphlan diphyly and the monophyly of Eulipotyphla. More surprisingly, we find strong evidence for a sister-group relationship between shrews and hedgehogs to the exclusion of moles.     

Congruent mammalian trees from mitochondrial and nuclear genes using Bayesian methods

Analyses of mitochondrial and nuclear gene sequences have often produced different mammalian tree topologies, undermining confidence in the merit of molecular approaches with respect to "traditional" morphological classification. The recent sequencing of the complete mitochondrial genomes of two additional rodents (Spalax judaei and Jaculus jaculus) and one lagomorph (Ochotona princeps) has prompted us to reinvestigate the issue. Using Bayesian phylogenetics, we found phylogenetic relationships between mammalian species highly congruent with previous results based on nuclear genes. Our results show the existence of four primary lineages of placental mammals: Xenarthra, Afrotheria, Laurasiatheria, and Euarchontoglires. Relationships between and within these lineages strongly suggest that the gene trees may also be congruent with the underlying species phylogeny.     

Nuclear gene sequences confirm an ancient link between New Zealand's short-tailed bat and South American noctilionoid bats

Molecular and morphological hypotheses disagree on the phylogenetic position of New Zealand's short-tailed bat Mystacina tuberculata. Most morphological analyses place Mystacina in the superfamily Vespertilionoidea, whereas molecular studies unite Mystacina with the Neotropical noctilionoids and imply a shared Gondwanan history. To date, competing hypotheses for the placement of Mystacina have not been addressed with a large concatenation of nuclear protein sequences. We investigated this problem using 7.1kb of nuclear sequence data that included segments from five nuclear protein-coding genes for representatives of 14 bat families and six laurasiatherian outgroups. We employed the Thorne/Kishino method of molecular dating, allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution on different branches on the phylogenetic tree, to estimate basal divergence times within key chiropteran clades. Maximum likelihood, minimum evolution, maximum parsimony, and Bayesian posterior probabilities all provide robust support for the association of Mystacina with the South American noctilionoids. The basal divergence within Chiroptera was estimated at 67mya and the mystacinid/noctilionoid split was calculated at 47mya. Although the mystacinid lineage is too young to have originated in New Zealand before it split from the other Gondwanan landmasses (80mya), the exact geographic origin of these lineages is still uncertain and will not be answered until more fossils are found. It is most probable that Mystacina dispersed from Australia to New Zealand while other noctilionoid bats either remained in or dispersed to South America.     

Chromosome painting in the manatee supports Afrotheria and Paenungulata

Background  

Sirenia (manatees, dugongs and Stellar's sea cow) have no evolutionary relationship with other marine mammals, despite similarities in adaptations and body shape. Recent phylogenomic results place Sirenia in Afrotheria and with elephants and rock hyraxes in Paenungulata. Sirenia and Hyracoidea are the two afrotherian orders as yet unstudied by comparative molecular cytogenetics. Here we report on the chromosome painting of the Florida manatee.     

Ancient SINEs from African endemic mammals

Afrotheria is a newly recognized taxon comprising elephants, hyraxes, sea cows, aardvarks, golden moles, tenrecs, and elephant shrews, each of which originated in Africa. Although some members of this taxon were once classified into distantly related groups, recent molecular studies have demonstrated their close relationships. It was suggested that this group emerged as a result of physical isolation of the African continent during the successive breakup events of Gondowanaland. In this study, a novel family of SINEs, designated AfroSINEs, was isolated and characterized from the genomes of afrotherians. This SINE family is distributed exclusively among the afrotherian species, confirming their monophyletic relationships. Furthermore, a distinct subfamily, which shares a deletion in the middle region of the SINE, was identified. The distribution of this subfamily is apparently restricted to the genomes of hyraxes, elephants, and sea cows, suggesting monophyly of these three groups, which was previously proposed as Paenungulata. We characterized the structures of the AfroSINEs from all afrotherian representatives by PCR, and we discuss how they were generated as well as the phylogenetic relationships of their host species.     

Ground plan and evolution of pterothoracic musculature of moths and butterflies (lepidoptera)

Investigation of the meso- and metathoracic musculature in 62 species of Lepidoptera revealed 40 topographically different muscles, 37 of which presumably belong to the ground plan. The archaic (homoneuran) taxa demonstrate a higher similarity in the musculature of the meso- and metathorax than more advanced (heteroneuran) taxa do. The number of muscles increases with body size but does not depend on the forewing shape or the surface area ratio of the hind and fore wings. Reduction of many muscles occurred repeatedly thus causing similarity between phylogenetically distinct lineages; development of new muscles is rare and occurs mostly as a result of splitting of a muscle into morphologically different parts. Decrease in the number of muscles is accompanied by an increase in their size, more advanced groups generally having relatively larger muscles, especially in the mesothorax. The thoracic anatomy of Lepidoptera is strongly linked with biomechanics of flight and therefore has a relatively low value for phylogenetic analysis, although some characters may be used in reconstruction of phylogeny at the level of subfamilies to superfamilies.