Phylogenetic inference using ND2 sequence data.

<p>Maximum-likelihood tree inferred from ND2 sequence alignment of the formalin-fixed sample (MVZ 214979), the Anocar2.0 reference genome (Anocar2.0), four <i>Anolis carolinensis</i> collected from Louisiana, USA, eight other <i>Anolis</i> species, and <i>Oplurus cyclurus</i>. <i>A</i>. <i>carolinensis</i> image printed under a CC BY license with permission of the original photographer and copyright owner J. Losos.</p

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing

<div><p>For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.</p></div

Nuclear coverage.

<p>(a) Read count and depth in 10 Kbp bins along the length of the six largest <i>A</i>. <i>carolinensis</i> chromosomes (green bars) using the MVZ 214979 library. The green line indicates a read count of 100, and coverage of 1X. (b) Read count and depth is shown in 1 Kb bins along a randomly selected 5 Mbp segment of chromosome 1 using the MVZ 214979 library.</p

Patterns of mismatches in MVZ 214979 sequences.

<p>The frequencies of the 12 types of mismatches (y-axis) are plotted as a function of distance from the 5′ and 3′ ends of the sequence reads (x-axis). The frequencies of each mismatch type are coded in different colors and line patterns. ‘After cleaning’ shows mismatch frequencies after deleting the first 50 bp form the end of each read.</p

Coverage of the mitochondrial genome.

<p>Distribution of read counts (in 10 bp bins) and depth of the mitochondrial genome from the MVZ 214979. A vertebrate mitochondrial map is used for reference on the bottom to label regions of protein coding and rRNA genes. The control region is at the end of the map and is not labelled.</p

A Novel Reproductive Mode in Frogs: A New Species of Fanged Frog with Internal Fertilization and Birth of Tadpoles

<div><p>We describe a new species of fanged frog (<i>Limnonectes larvaepartus</i>) that is unique among anurans in having both internal fertilization and birth of tadpoles. The new species is endemic to Sulawesi Island, Indonesia. This is the fourth valid species of <i>Limnonectes</i> described from Sulawesi despite that the radiation includes at least 15 species and possibly many more. Fewer than a dozen of the 6455 species of frogs in the world are known to have internal fertilization, and of these, all but the new species either deposit fertilized eggs or give birth to froglets.</p></div

Distribution map (left panel) depicting the range of <i>Limnonectes larvaepartus</i>.

<p>The right panel shows the phylogenetic position of <i>L. larvaepartus</i>, with different symbol shapes denoting regional genetic structure in the species.</p

Images of <i>Limnonectes larvaepartus</i>.

<p>(<i>a</i>) MVZ 268323 (<b>male, left</b>) and MVZ 268307 (<b>female, right</b>) collected from Desa Uaemate along the Tasio-Tibo Road, Kabupatan Mamuju, Provinsi Sulawesi Barat, Sulawesi Island (02.61287S, 119.14238 E, 89 m elev.); (<i>b</i>) <i>Limnonectes larvaepartus</i> female (MVZ 268426) with tadpoles removed from the oviduct. Note the large yolk reserves available to the tadpoles; (<i>c</i>) An <i>in situ</i> adult male <i>L. larvaepartus</i> (JAM 14234) observed calling while perched on the edge of a small pool 2 m away from a 2 m wide stream; several <i>L. larvaepartus</i> tadpoles were present in the pool including the two visible within the yellow circle; (<i>d</i>) dorsal and ventral views of ∼stage 25 <i>L. larvaepartus</i> tadpoles (JAM 14271) released by a pregnant female (JAM 14237) at the moment of capture.</p

Summary of univariate morphological variation among mensural characters in <i>Limnonectes larvaepartus</i>.

<p>Summary of univariate morphological variation among mensural characters in <i>Limnonectes larvaepartus</i>.</p

Dmelanostictus_WoganetalBioLetters

nexus file of 3 concatenated gene