Implications for Bat Evolution from Two New Complete Mitochondrial Genomes

Yu-Hsin LinGo, and David Penny

Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand

Mitochondrial genomes are useful in the quantitative analysis of vertebrate evolution. We report here the complete mitochondrial genomes for a megabat (the flying fox, Pteropus scapulatus) and a microbat (the New Zealand long-tailed bat, Chalinolobus tuberculatus). The evolutionary history of bats (chiroptera) has been uncertain, and even the monophyly of this group has been questioned. The new sequences allow five questions to be addressed: the position of bats within eutheria, whether bats are monophyletic, whether microbats are paraphyletic with respect to megabats, the approximate timing of the origin of bats, and whether some insectivores (e.g., moles) form a sister group with bats. In order to examine these questions, we analyzed two data sets (both separately and combined), one with 12 protein-coding regions and the other with RNA (combined ribosomal RNAs and tRNAs). The results are congruent, support bat monophyly, and place bats close to the cetferungulates (whales [cetaceans] plus ferungulates [carnivores, ungulates, and perissodactyls]).

The position of bats in the eutherian tree is uncertain. The most common assumption from anatomical evidence has been that they group with primates, flying lemurs, and tree shrews, forming the Archonta (Szalay 1977Citation ; Novacek 1992). However, in a phylogenetic analysis of the complete mitochondrial genome of the Jamaican fruit bat (Artibus jamaicensis; Pumo et al. 1998Citation ) it appeared more closely related to cetferungulates, a clade including Cetacea, Artiodactyla, Perissodactyla, and Carnivora. Phylogenetic analysis from the c-myc gene sequences also support this relationship (Miyamoto, Porter, and Goodman 2000). The recent publication of the mitochondrial genome of the mole (Talpa europaea; Mouchaty et al. 2000) placed this insectivore close to the Jamaican fruit bat. The sister position of the mole and the Jamaican fruit bat can be further tested by the addition of our two new bat mitochondrial genomes.

Even the relationship between microbats and megabats is uncertain. On the basis of neuroanatomy, it has been suggested that megabats are more closely related to primates than to microbats (Pettigrew 1986Citation ; Pettigrew et al. 1989Citation ). This would imply a diphyletic origin for bats, a hypothesis which has not been supported by sequence data (Bennett et al. 1988Citation ; Mindell, Dick, and Baker 1991Citation ; Van Den Bussche et al. 1998Citation ) and can now be tested with much longer sequences, such as complete mitochondrial genomes. In contrast to the hypothesis of bat diphyly, it has recently been suggested (Hutcheon, Kirsch, and Pettigrew 1998Citation ; Teeling et al. 2000Citation ) that megabats are derived from within microbats. In particular, megabats are suggested to be closer to rhinolophid microbats. Thus, megabats would be strictly monophyletic, but microbats would be paraphyletic (with megabats arising within microbats). However, on this hypothesis the two microbat mitochondrial genomes available (see below) are still expected to be monophyletic; it is this aspect that is tested here.

It is also necessary to get good estimates of the timing of divergence of the main eutherian lineages, particularly to estimate how many mammals survived from the Cretaceous to the Tertiary (Hedges et al. 1996Citation ; Cooper and Penny 1997Citation ; Penny and Hasegawa 1997Citation ). A reliable evolutionary tree is required before good estimates of timing are possible. Bats are one of the best candidates for breaking the K-T crown group barrier (Waddell, Okada, and Hasegawa 1999Citation ). The two bat mitochondrial genomes reported here help address all five questions.

The New Zealand long-tailed bat is a member of the largest bat family (Vespertilionidae) and on morphology is closely related to five Australian species. It is assumed that this group evolved in Australia and that C. tuberculatus crossed the Tasman Sea to New Zealand about 1 MYA (Daniel 1990Citation ). Because of their low numbers, they are a threatened species. The other microbat for which a complete mitochondrial genome is already available is the Jamaican fruit bat in the family Phyllostomidae (Pumo et al. 1998Citation ). The relatively distant relationship between these two microbats (Jamaican fruit bat and New Zealand long-tailed bat) is expected to help stabilize the phylogenetic tree and decrease long- branch effects (Hendy and Penny 1989Citation ). However, these two microbats are expected to be monophyletic; it is the rhinolophid microbats that are suggested to be deeper in the tree than megabats (Teeling et al. 2000Citation ).

Genomic DNA of flying fox and long-tailed bat was amplified in fragments longer than 5 kb (in order to avoid amplifying nuclear copies) using the Expand Long Template PCR kit (Roche). Long PCR DNA fragments were sequenced directly and also used as template for a second short-range PCR 0.5~1.5 kb. Sequencing reactions were done according to standard protocols and run on the 377 ABI Applied Biosystems automated DNA sequencer at the Massey University Sequencing Facility. Because we were sequencing several complete mtDNA genomes, we designed most primers from conserved regions of the mtDNA genomes of mammals and birds and allowed 0–5 degenerate sites to maximize their usefulness for other species. We used the Fasta search in the GCG program (Wisconsin Package, version 10.0) to search our primer database for appropriate targets for primer walking. When none were available, new primers were designed using Oligo 4.03 (National Biosciences, Inc.). Sequences were checked and assembled using the Sequencing Analysis and Sequence Navigator programs (ABI).

Complete mammalian mtDNA sequences were obtained from GenBank for the following 23 taxa: mouse Mus musculus (NC_001569); rat Rattus norvegicus (NC_001665); rabbit Oryctolagus cuniculus (NC_001913); guinea pig Cavia porcellus (NC_000884); dormouse Myoxus glis (NC_001892); human Homo sapiens (NC_001807); baboon Papio hamadryas (NC_001992); Gibbon Hylobates lar (NC_002082); aardvark Orycteropus afer (NC_002078); armadillo Dasypus novemcinctus (NC_001821); fruit bat Artibeus jamaicensis (NC_002009); dog Canis familiaris (NC_002008); cat Felis catus (NC_001700); harbor seal Phoca vitulina (NC_001325); horse Equus caballus (NC_001640); hippopotamus Hippopotamus amphibius (NC_000889); cow Bos taurus (NC_001567); sheep Ovis aries (NC_001941); white rhinoceros Ceratotherium simum (NC_001808); fin whale Balaenoptera physalus (NC_001321); mole Talpa europaea (NC_002391); pig Sus scrofa (NC_000845); and elephant Loxodonta africana (NC_000934).

In order to compare results quantitatively, we prepared three data sets; the first contained RNA sequences (rRNAs and tRNAs), the second contained 12 protein genes coded on the same DNA strand, and the third combined both data sets. Sequences for the 25 species were aligned manually in SeAl, version 1.0a1 (http://evolve.zps.ox.ac.uk/Se-Al/Se-Al.html). The rRNA sequences were aligned with reference to the secondary structure (http://www.rna.icmb.utexas.edu/RNA/) to maximize homologous positions. The RNA alignment data set can be obtained from the MBE web site. There were no difficulties aligning the proteins. PAUP*, version 4.0b3a (Swofford 1998Citation ), was used for all data sets. MOLPHY (Adachi and Hasegawa 1996Citation ) was used for a maximum-likelihood analysis of amino acid sequences.

Our two new complete mitochondrial genomes are available from GenBank (accession numbers AF321050 and AF321051). More than 90% of both genomes were sequenced in both directions. The two sequences have the standard gene order of mammals and are 16,818 and 16,741 nt long for flying fox and long-tailed bats, respectively. Both bats have a 6-bp repeat (CATACG) at the end of the D-loop, which occurs more than 30 times. Apart from this, the genomes do not show any unusual features.

The results are dependent on the tree-building methods and data sets used and are summarized in table 1 and figure 1A. In addition to constructing trees from the DNA sequences, we checked the conclusions using Lento plots (fig. 1B and C ) based on the inferred amino acid sequences using LogDet method in the ProtDet program (Penny et al. 1999Citation ). The monophyly of bats is supported in our study using all data sets and different analysis methods. However, we do not find universal support for the two microbats as sister taxa. The 6-bp repeat found in the control region of the New Zealand long-tailed bat and the flying fox is not found in the Jamaican fruit bat. This implies either that it is ancestral in bats and has been lost (on this branch) or that it is independently derived in both the flying fox and the New Zealand long-tailed bat. Microbats from Rhinolophoidea are predicted to be more closely related to megabats than they are to microbats (Teeling et al. 2000Citation ), but even if the microbats form a monophyletic clade, the distance between the microbat and the megabat will be quite small.


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Table 1 Comparative Results for Seven Hypotheses

 


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  Fig. 1.—A, Phylogenetic tree of three bats with other eutherian mammals. The tree was inferred by maximum-likelihood analysis of combined protein-coding and RNA sequences. The two arrows with dotted lines show the alternative rearrangements for the mole (options E and G in table 1 ); the two arrows with solid lines show the alternative rearrangements of the three bats (options C and D in table 1 ). B, A Lento plot of support and conflict for the main partitions (splits) with 22 taxa from A (excluding the armadillo, the aardvark, and the elephant). Values are calculated from LogDet distances on amino acids. Partitions are ranked according to their support, with constant sites downweighted by half (see below). Partitions (signals) in the closest tree are indicated by solid bars in the support (above x-axis) and by open bars in contradiction of partitions (below x-axis). The 14 strongest partitions are in the closest tree. The correspondence between the signals in the Lento plot and the tree in A are indicated by the numbers 1–18. Numbers 15 and 16 are not in the tree in A. The closest tree with LogDet distances from amino acids gives option G of table 1 . C, The support for eight partitions as constant sites are progressively removed (downweighted). The 11 successive values are from constant sites given full weight (1.0 on the x-axis) to sites given 10% of full weight (0.1 on x- axis). The three partitions at the front of the top figure of C are for the three subtrees joining the three bats (options D, C, and B in table 1 ). In this analysis, the monophyly of the two microbats is supported, irrespective of the weighting of constant sites. In the bottom figure of C, the three front signals are for bats closer to ferungulates than moles (option F in table 1 ), bats and moles forming a sister group (option E), and moles closer to ferungulates (option G). In this case, the support for option E increases as constant sites are downweighted, and support for option G decreases. Although this is just a local rearrangement on the tree, more data are required to stabilize this local change. The two signals at the back are for the monophyly of bats (upper figure) and for the monophyly of bats and moles plus ferungulates (bottom figure). Thus, both aspects of the tree are independent of unequal amino acid composition (because of LogDet) and of the exact numbers of sites that are genuinely invariant

 
Thus, the Archonta (primates, tree shrews, flying lemurs, and bats) did not form a natural group in our analyses. Our analyses support those of Pumo et al. (1998), who propose the grouping of cetferungulates as a clade, with bats as a sister group (fig. 1 ). A recently published mitochondrial genome for a tree shrew (Tupaia belangeri; Schmitz, Ohme, and Zischler 2000) was included later in our data set; our results confirmed those of Schmitz, Ohme, and Zischler (2000), that bats and tupaia are not closely related (results not shown).

These results have implications for the understanding of bat evolution, for example, flight in bats may have arisen only once within the Chiroptera (see also Allard, McNiff, and Miyamoto 1996Citation ). Our results also suggest that the different visual systems of microbats and megabats do not reflect a paraphyletic origin of bats, but may be a recent adaptation for their respective environments. Hence, "brain evolution can be rapid and that even closely related species can have quite different visual systems" (Kaas and Preuss 1993Citation ).

The direct association of moles with bats in earlier analysis (Mouchaty et al. 2000) could be a long-branch attraction artifact. The position of moles is locally stable (Cooper and Penny 1997Citation ) in the eutherian tree (fig. 1 and table 1 ). Most of our trees put moles as a sister group to a clade containing cetferungulates and bats. However, occasionally moles are nested inside cetferungulates or joined with bats. Removing our two bats causes moles to join with Jamaican fruit bats with high bootstrap support, as mentioned in Mouchaty et al. (2000). If either of our bat sequences are included, moles do not join with Jamaican fruit bats. Thus, it is possible that the long branch of moles was attracted to Jamaican fruit bats. The best way to stabilize the position of moles is probably by adding more taxa from Lipotyphla (e.g., shrews) to the analysis.

The oldest bat fossil appears in deposits of the Eocene epoch about 50 MYA. Because these fossil bats are "typical" bats, they do not give any clues about which mammals gave rise to the Chiroptera (Altringham 1996Citation ). The origin of the order Chiroptera therefore must be much older than 50 Myr. An analysis including the new mitochondrial genome of sperm whales (Arnason et al. 2000) estimated the time of divergence of cetferungulata and moles/bats at 69.5–78.2 MYA based on three different calibration points. Our results clarify that moles and bats are not in a clade and the divergence time of bats with cetferungulata will be at least 70–78 MYA, which is well before the K-T boundary. However, before the times are reinvestigated, it is preferable to have additional insectivore taxa—a stable and correctly rooted tree is essential for good divergence times. After we have a stable bat/eulipotyphla tree, we will reestimate the timing of the bat divergences.

It is interesting that with the dormouse genome included, the four rodents come together under maximum likelihood, although the position of rabbits is less stable. With some methods, rabbits do not join with rodents to form glires. A relative of rabbits, the pika, is a good candidate to stabilize the position of rabbits. As part of the current project, we are sequencing mitochondrial genomes of a pika (Ochotona collaris), as well as those of a shrew (Soriculus fumidus), a gymnure (Echinosorex gymnurus), and another bat (Rhinolophus monoceros), in order to help clarify all the problems mentioned above.

With respect to the five questions posed in the introduction, our conclusions are as follows: Bats (micro and mega) form a monophyletic group. There is strong support for cetferungulates (not primates) being the most closely related sister group to bats. The grouping of Vespertilionidae and Phyllostomidae is as expected and places limits on the paraphyly of microbats, although at least 14 other bat families await examination. The approximate time of the origin of bats is >70 MYA, and, finally, the position of moles is locally stable around cetferungulates plus bats. The conclusions are reinforced by the similarity of the trees from the two independent data sets, containing RNA and protein-coding regions, respectively.Note Added in ProofNikaido et al. (2000) have recently published the mitochondrial genome for a different flying fox (J. Mol. Evol. 51:318–328). They come to the same conclusion with respect to bat monophyly.

Acknowledgements

We thank Jack Pettigrew of Queensland University, Brisbane, Australia, for providing flying fox DNA; Brian Lloyd of the Department of Conservation, Wellington, New Zealand, for long-tailed bat tissue samples; and Abby Harrison and Matthew Phillips for running the laboratory smoothly and help in aligning and analysis. The New Zealand Marsden Fund supported this work.

Footnotes

Ross Crozier, Reviewing Editor

1 Keywords: bat evolution long branch attraction mammal phylogeny mitochondrial genomes Back

2 Address for correspondence and reprints: Yu-Hsin Lin, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand. y.lin{at}massey.ac.nz Back

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Accepted for publication December 12, 2000.