trigoniulus corallinus , sometimes called the rusty millipede , is a species of millipede widely distributed in the indo-malayan region including india , sri lanka , china , myanmar , thailand , vietnam , malaysia , singapore , and much of indonesia .

it is also reported from fiji and tanzania and found in south asia and the caribbean as an introduced species .

these millipedes inhabit moist areas , rotten wood and compost .

the genome of t. corallinus was sequenced in 2015 , the first time this has been done for a millipede .

trigoniulus corallinus

genome of the rusty millipede , trigoniulus corallinus , illuminates diplopod , myriapod , and arthropod evolution .
genome of the rusty millipede , trigoniulus corallinus , illuminates diplopod , myriapod , and arthropod evolution . - pubmed - ncbi
trigoniulus corallinus biology . ( a ) phylogenetic relationships of diplopods and related clades . phylogeny based on regier et al . ( 2010 ) ( not all clades shown ) . ( b ) trigoniulus corallinus distribution . native range indicated broadly ( blue ) , with known introduced populations indicated in yellow . note that introduced t . corallinus may well be found elsewhere . ( c ) adult t . corallinus , approximately 5 cm in length . ( d ) egg capsules gathered in captivity as described in materials and methods . ( e ) egg case dissected to show single egg . in both ( d ) and ( e ) , white scale bar represents 1 mm in length . ( f ) overview of genomic dna sequencing , genome assembly procedures , and final data figures from this study .
rowland m . shelley , robert m . carmany & joseph burgess ( 2006 ) .
introduction of the milliped , trigoniulus corallinus ( gervais , 1847 ) ( spirobolida : trigonuilidae ) , in florida , u . s . a .
. entomological news 117 ( 2 ) : 239–241 .
trigoniulus corallinus has much potential as a model species . although the centipede s . maritima has an already well - developed community and history as a scientific model , we suggest that t . corallinus may be of broader utility as a myriapod model organism . it is a cosmopolitan species available commercially when not collectible in the wild . unlike s . maritima it can be kept in the laboratory and will breed in controllable conditions . although protocols for its use in a variety of developmental contexts remain to be established , there is no reason why this species could not become an important system for developmental biology research .
information from a diplopod outgroup is of wide utility for the discernment of ancestral arthropod characters when compared with better known insect model organisms and new crustacean models , such as the water flea daphnia and shrimp neocaridina ( colbourne et al . 2011 ; kenny et al . 2014 ) . furthermore , the cosmopolitanly distributed diplopod t . corallinus is easily cultivable and will breed in captivity , providing a ready resource for embryological study . trigoniulus corallinus therefore represents a potentially intriguing model for future work in developmental , ecological and evolutionary spheres of scientific investigation , and the initial genomic resources presented here will be of great interest to a variety of fields .
myriapod mtdna interrelationships : tree recovered by bayesian and maximum - likelihood inference of myriapod and panartropod interrelationships , on the basis of concatenated coding nucleotide sequences of 13 protein - coding genes , performed as described in materials and methods . trigoniulus corallinus boxed in red for ease of identification . familial , order , and class level classifications shown at right . note : symphalans are positioned as sister taxa to all other myriapods , in marked contrast to established phylogenies . bootstrap proportions ( as percentage , 1 , 000 replicates ) and bayesian posterior probabilities ( maximum 1 . 00 ) shown at base of nodes . scale bar represents sequence changes per site at unit distance .
mitochondrial genome gene order across the myriapoda , as compared with panarthropod and hexapod ground patterns , with that of t . corallinus boxed in red . note similarities to n . annularus , also a member of the spirobollidae . genes colored for ease of recognition .
( of iulus corallinus eydoux & souleyet , 1842 ) gerstäcker , a . ( 1873 ) . die gliederthier - fauna des sansibar - gebietes . 1 - 542 . leipzig , heidelberg page ( s ) : 516 ; note : spirobolus lumbricinus , syn by shelley , 1999 [ details ]
( of iulus corallinus eydoux & souleyet , 1842 ) gervais , p . ( 1847 ) . myriapodes . in : walckenaer , hist . natur . des insectes . aptères iv , 4 : 1 - 623 . paris , available online at urltoken page ( s ) : 171 [ details ]
( of iulus corallinus eydoux & souleyet , 1842 ) hoffman , r . l . ( 1999 ) . checklist of the millipedes of north and middle america . virginia museum of natural history , special publication , 8 : 1 - 584 . martinsville page ( s ) : 103 [ details ]
novel mycin sequences : alignment of known mycin ( antifungal ) genes from d . melanogaster , caenhorhabditis remanei , and novel mycins from t . corallinus . alignment visualized in jalview , colored with clustalx identity . note regions of high conservation around shared cysteine residues , with more divergence elsewhere , particularly at the n terminus of these sequences .
( of iulus corallinus eydoux & souleyet , 1842 ) pocock , r . i . ( 1888 ) . contribution to our knowledge of the myriapoda of dominica . annals and magazine of natural history , ser . 6 , 2 page ( s ) : 481 ; note : spirobolus dominicae , syn by shelley , 1999 [ details ]
( of iulus corallinus eydoux & souleyet , 1842 ) porat , c . o . von ( 1876 ) . om några exotiska myriopoder . kongl . svenska vetenskaps - akademiens handlingar , bihang , 4 ( 7 ) : 3 - 48 . stockholm page ( s ) : 136 ; note : spirobolus göesi , syn by shelley , 1999 [ details ]
( of iulus corallinus eydoux & souleyet , 1842 ) enghoff , h . ; golovatch , s . i . ; nguyen duc , a . ( 2004 ) . a review of the millipede fauna of vietnam ( diplopoda ) . arthropoda selecta , 13 ( 1 - 2 ) : 29 - 43 . moscow page ( s ) : 35 [ details ]
the organization of the t . corallinus ( myriapoda : trigoniulidae ) mitochondrial genome . orientation of genes ( transcription clockwise or anticlockwise represented outside or inside the form , respectively ) is represented by the outside circle . local gc content , ( gc dark blue , at light blue ) represented on the inner ring . image displayed in organellargenomedraw ( ) . photograph by the authors .
( of iulus corallinus eydoux & souleyet , 1842 ) bollman , c . h . ( 1888 ) . description of a new species of insect , fontaria pulchella , from strawberry plains , jefferson county , tenn . proceedings of the united states national museum , 11 : 316 page ( s ) : 214 ; note : spirobolus sanctae - luciae , syn by shelley , 1999 [ details ]
the organization of the t . corallinus ( myriapoda : trigoniulidae ) mitochondrial genome . orientation of genes ( transcription clockwise or anticlockwise represented outside or inside the form , respectively ) is represented by the outside circle . local gc content , ( gc dark blue , at light blue ) represented on the inner ring . image displayed in organellargenomedraw ( lohse et al . 2007 ) . photograph by the authors .
( of iulus corallinus eydoux & souleyet , 1842 ) koch , c . l . ( 1847 ) . system der myriapoden mit den verzeichnissen und berichtigungen zu deutschlands crustaceen , myriapoden und arachniden . in : panzer & herrich - schäffer , a . : kritische revision der insectenfaune deutschlands , iii . bändchen , regensburg , 1 - 196 . regensburg , available online at urltoken page ( s ) : 202 [ details ]
myriapods have been previously noted as displaying diversity in mtdna sequence and gene structure ( e . g . , gai et al . 2008 ; lavrov et al . 2002 ) , and the sequencing of the t . corallinus genome provided an ideal opportunity to add to our presently poor sampling of myriapod mtdna sequences—as of october 28 , 2014 , 14 myriapod mtdna sequences were available in public sources , a fraction of the 13 , 000 extant species of this clade .
in some ways the terrestrial lifestyle of t . corallinus suggests that it may possess more ancestrally shared characters than s . maritima , given the subterranean , marine lifestyle of the latter species . this is borne out by investigation of known gene families , whose absence in s . maritima could not previously be discerned to have resulted from selective pressure in the geophilomorph centipedes or ancestral loss . perhaps unsurprisingly , given the presence of classical ocelli in this species , canonical opsin genes are found in this genome . classical circadian clock - driving proteins , such as period ( per ) , clock ( clk ) and cycle ( cyc ) , are all found in the t . corallinus genome ( supplementary file s1 , supplementary material online ) , further reinforcing the lineage - specific nature of loss in the s . maritima resource ( chipman et al . 2014 ) . the presence of such genes robustly underlines the utility of a diplopod genomic resource .
( of iulus corallinus eydoux & souleyet , 1842 ) hoffman , r . l . ; golovatch , s . i . ; adis , j . ; demorais , j . w . ( 2002 ) . 5 . 2 diplopoda . adis , joachim [ ed . ] . amazonian arachnida and myriapoda : identification keys to all classes , orders , families , some genera , and lists of known terrestrial species . pensoft : 505 - 533 , 505 - 533 . sofia , moscow page ( s ) : 533 [ details ]
cegma ( parra et al . 2007 , 2009 ) results showing coverage of the expected metazoan gene complement are excellent—171 / 248 ultraconserved core eukaryotic genes ( cegs ) are recovered as present in our data ( 68 . 95 % ) . from blast results ( tblastn , e < 10 − 9 ) , 432 / 458 kog groups ( 94 . 3 % ) were shown to be present as noted in supplementary file s1 , supplementary material online , with 349 / 458 ( 76 . 2 % ) supported by all six species used by cegma . the missing 26 eukaryotic orthologous groups ( kog ) s groups are listed in supplementary file s1 , supplementary material online . as the genome of s . maritima contained 95 . 1 % ( cf . t . corallinus 94 . 3 % ) of this data set , we suggest that we have recovered the majority of coding sequence in our assembly , albeit at low contiguity . furthermore , taken together with the results of our targeted investigations into specific gene families , detailed below , we find no evidence for large - scale gene loss in the t . corallinus genome .
the utility of myriapod mitochondrial genomes for the reconstruction of arthropod phylogenetics has been the subject of some recent debate , due to the extensive rearrangement seen in the myriapod taxa ( brewer et al . 2013 ) . the t . corallinus mitochondrial genome appears similar in general arrangement to that of other related millipedes , and particularly that of the only other spirobolid mitochondrial genome yet sequenced , that of n . annularis . by other metrics however , such as at % , the t . corallinus genome is quite different to even that of its closest sequenced relative . these differences have obfuscated phylogenetic inference using mtdna sequences in the myriapoda in the past ( e . g . , brewer et al . 2013 ) and this was also found to an extent in our data , with symphalans posited as the sister group of all other myriapods , a likely artifact , perhaps caused by high rates of change in this clade . further sampling of myriapod mtdna is necessary to unravel the complex and intriguing patterns of evolution seen in these organelles in this clade ( lavrov et al . 2000 , 2002 ; brewer et al . 2013 ) , and our data will be a vital addition to this effort .
other than the hoxl class , our data set also offers interesting insights . as in the centipede genome , a clear dmbx ortholog is identified in the genome of t . corallinus , whose sequence can be found in supplementary file s1 , supplementary material online . its presence in myriapods and thus representatives of all bilaterian superphyla confirms further the presence of this gene at the base of the bilaterian radiation , and , along with its presence in annelids ( takahashi and holland 2004 ; kenny and shimeld 2012 ) suggests a means by which the origin of its role in demarcating the midbrain / hindbrain boundary may be tested .
it was noted in investigations of the s . maritima genome that duplication / paralogs were used to generate coding sequence diversity , where insects use alternate splicing ( chipman et al . 2014 ) . to investigate whether this trait is observed more broadly in the myriapoda , we have assayed cases noted from that species in the t . corallinus genome . the most pronounced example of duplication observed in the centipede was the down syndrome cell adhesion molecule ( dscam ) gene family , where over 100 unique loci were noted ( chipman et al . 2014 ) . using blast to compare s . maritima dscam homologs to our data set ( tblastn , cutoff 10 − 9 ; supplementary file s1 , supplementary material online ) , we recover 43 contigs containing the dscam ig7 domain .
however , care should be taken before assuming duplicates to be present ancestrally on the basis of duplicate loci in s . maritima , as duplications observed in that species are not necessarily shared across all myriapods . for instance , a single copy of gene cap’n’collar ( cnc ) has also been noted as exhibiting paralogy rather than splice variation in s . maritima ( nucleotide and amino acid sequence ; supplementary file s1 , supplementary material online ) , but only a single example is found in t . corallinus . although proving absence is difficult , the clear homology of this gene to its orthologs and our generally high recovery of coding regions of the genome gives us confidence that a second paralog would be spotted if present . the duplication of cnc in s . maritima therefore seems to be specific to that species , rather than a myriapod symplesiomorphy .
the recently published s . maritima genome data set was distinguished by its high levels of conservation across the majority of characterized gene families ( chipman et al . 2014 ) . to further test whether this “prototypical arthropod cassette” was also found in the millipede genome , we compared the complete annotated s . maritima proteome to the t . corallinus genome . of the 26 , 950 peptides in the drosophila melanogaster genome ( bdgp5 . 23 release ) , 15 , 995 ( 59 . 4 % ) possess a putative ortholog in the millipede genome ( tblastn , e < 10 − 9 ) . this is comparable to the number of s . maritima genes possessing a hit in the d . melanogaster proteome . of 15 , 008 s . maritima peptides ( release 23 , downloaded from ensembl , september 18 , 2014 ) , 9 , 168 ( 61 . 1 % ) possess a putative ortholog in d . melanogaster ( blastp , e < 10 − 9 ) .
introduction the rusty millipede ( trignoiulus corallinus ) is a medium to large - sized millipede commonly found in central taiwan ( and in other areas as well ) . the body is brick red in color with pale black bands on the sides . the adult grows up to 5cm in length and can often be found in dead leaves and low rock walls around the botanical garden at dawn or in the evening . individuals are also frequently sighted crossing paved paths . their relatively large size means they are easy to see so squashed rusty millipedes are a common sight in the morning . the rusty millipede however is quite harmless and is in fact a beneficial insect that helps break down dead leaves into organic soil for plants . the rusty millipede usually breeds in summer around july and august . during this time , they can often be found mating in shady areas near rocks or stone walls at dawn or in the evening . though not rare , the species was only formally recorded and classified in taiwan by dr . zoltan korsos in 2004 .
the exoskeleton of arthropods is made up of the polysaccharide chitin , along with a range of cuticular proteins . many kinds of cuticular protein are known , but the cpr family is the most common , with up to 150 genes found in some species of arthropod ( willis 2010 ) . these proteins can be recognized by a conserved , approximately 64 amino acid sequence ( rebers and willis 2001 ) . searches using sequences of known orthology of our data set identified 26 instances of this sequence in the t . corallinus genome . this is slightly fewer than the 39 members identified in the s . maritima genome ( chipman et al . 2014 ) , and as in that data set , both rr - 1 ( flexible cuticle ) and rr - 2 ( rigid cuticle ) associated forms of these genes can be identified , entirely consistent with the mandibulate origin of the rr - 1 family as proposed in chipman et al . ( 2014 ) . the nucleotide and amino acid sequences of these putatively identified domains can be found in supplementary file s1 , supplementary material online .
again similarly to observations in s . maritima , no odorant receptor ( or ) genes could be identified in our data set using a variety of sequences of known orthology at lenient blast settings ( tblastn , e cutoff 1 ) . this is consistent with the findings of robertson et al . ( 2003 ) , who suggested the or family represents an insect novelty . surprisingly we were unable to identify many gustatory receptor ( gr ) genes in our data set , finding only two sequences with clear homology to this family ( sequences , supplementary file s1 , supplementary material online ) , a marked contrast with s . maritima , which possesses 77 of these genes . gr genes are known to have existed in the arthropod common ancestor , and have been observed in arachnids and crustaceans as well as in the centipede , but the marked diversification seen in s . maritima seems limited to that species . a relatively large number of ionotropic receptor ( ir ) genes can be found in the t . corallinus genome , although not as many as the 69 observed in s . maritima . a total of 23 ir sequences ( listed in supplementary file s1 , supplementary material online ) were found in our data set , of all ir classes . this restricted complement relative to the centipede may reflect the use of duplication to build diversity in s . maritima , as noted earlier in this article .
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kenny nj 1 , shen x 2 , chan tt 1 , wong nw 1 , chan tf 2 , chu kh 3 , lam hm 2 , hui jh 4 .
simon f . s . li marine science laboratory of school of life sciences and center of soybean research of state key laboratory of agrobiotechnology , the chinese university of hong kong , shatin , hong kong .
center of soybean research of state key laboratory of agrobiotechnology , the chinese university of hong kong , shatin , hong kong .
simon f . s . li marine science laboratory of school of life sciences , the chinese university of hong kong , shatin , hong kong .
simon f . s . li marine science laboratory of school of life sciences and center of soybean research of state key laboratory of agrobiotechnology , the chinese university of hong kong , shatin , hong kong jeromehui @ cuhk . edu . hk .
pmid : 25900922 pmcid : pmc4453065 doi : 10 . 1093 / gbe / evv070
nathan j . kenny , 1 xin shen , 2 thomas t . h . chan , 1 nicola w . y . wong , 1 ting fung chan , 2 ka hou chu , 3 hon - ming lam , 2 and jerome h . l . hui 1 , *
* corresponding author : e - mail : kh . ude . khuc @ iuhemorej .
data deposition : data has been deposited in ncbi databases ( bioproject prjna260872 , biosample samn03048671 , experiment srx700727 ) and made available for download as noted in text .
copyright © the author ( s ) 2015 . published by oxford university press on behalf of the society for molecular biology and evolution .
this is an open access article distributed under the terms of the creative commons attribution non - commercial license ( urltoken ) , which permits non - commercial re - use , distribution , and reproduction in any medium , provided the original work is properly cited . for commercial re - use , please contact journals . permissions @ urltoken
the myriapoda contains greater than 13 , 000 described species and is one of the most speciose metazoan subphyla . recent molecular analysis suggests that the myriapoda is the sister group to the pancrustacea (
) , limiting our ability to draw inferences into myriapod biology , as well as providing only a single outgroup from this clade for comparison to the pancrustacea .
millipedes can be found worldwide performing vital ecological roles as detritovores ( snyder et al . 2009 ; shelley and golovatch 2011 ) , and can be easily distinguished from other myriapods—segments posterior to the fourth from the head have two pairs of limb per segment ( diplosegments ) ( barnes 1982 ) . the oldest terrestrial metazoan fossil is that of a millipede ( pneumodesmus newmani ) dated at approximately 428 myr of age ( wilson and anderson 2004 ) . millipedes have the ability to synthesize a range of defensive chemical components to ward off predators ( shear et al . 2007 , 2010 ) , and a genomic resource will be useful to understand the metabolism of these organisms for a range of research into these novel pathways , which could represent a source for fungicides and other novel bioproducts ( roncadori et al . 1985 ) .
genomic dna was extracted from the organism after starvation using a dneasy blood & tissue kit ( qiagen ) following the manufacturer’s protocol , and sequenced on the illumina hiseq2000 platform by beijing genomics institute ( bgi ) hong kong .
for all analyses , the more complete ( 100 bp + ) data set was utilized . to determine coverage of the core eukaryotic gene cassette , cegma ( parra et al . 2007 ) was run with all default settings . for identification of other genes , ncbi - blast - 2 . 2 . 23 + ( altschul et al . 1990 ) tblastn searches were run using genes of known homology from the ncbi nr database as query sequences . the sequence of the contigs putatively identified was then reciprocally blasted ( blastx ) to the ncbi nr database for confirmation of identity .
gdna sample was sequenced on the illumina hiseq2000 platform by bgi hong kong . reads were provided from an external server , and a summary of these data can be seen in
. the data have been uploaded to ncbi’s short read archive ( bioproject prjna260872 , biosample samn03048671 , experiment srx700727 ) . read quality as assayed using fastqc was found to be good ( lower quartile phred score greater than 31 through to the 100th base for both read data sets ) , as can be observed in
) was selected for further optimization . initial assemblies were used to find expected coverage and average sequenced fragment size , and a final optimized assembly with settings as described in the materials and methods section was performed , resulting in 1 , 305 , 238 contigs with a size of 100 bp or above , before the removal of contaminating sequence .
initial analysis of our read data detected ribosomal gene sequence from non - millipede species . myriapods in general and
) . we therefore assayed our genomic resource for evidence of these species , using known ribosomal rna sequences . although we did not recover any evidence of
) , fragmentary ribosomal rna sequences with blast identity to a number of ciliophoran protist species were recovered in our data set before contamination removal . to ensure our data were not more broadly contaminated with protist sequence , all protist and bacterial genomic data present in ncbi databases as of september 16 , 2014 were used as the basis for comparison and removal using deconseq standalone 0 . 4 . 3 (
) . a total of 71 , 302 contigs were removed from our initial assembly ( 5 . 46 % ) . contigs removed will include shared repetitive elements , and as a result our data set should not be used for making conclusions about such regions of the genome . statistics for the final assembly after the removal of contamination , given for contigs 200 and 100 bp in size and greater , are presented in
of 15 , 008 proteins in the s . maritima genome , 8 , 459 ( 56 . 4 % ) possessed a putative ortholog in our data set ( tblastn , e < 10 − 9 ) . surprisingly , this was fractionally lower than d . melanogaster complement recovery . previous estimates have suggested that 32 % of genes present in the strigamia genome are the result of gene duplication events unique to the myriapoda lineage ( chipman et al . 2014 , phylomedb analysis ) , but the low percentage of centipede proteins recovered in our genome may mean that these genes are largely strigamia - or centipede - specific , rather than shared across the myriapoda .
we therefore suggest that myriapods share duplication at some loci , rather than utilizing splice variation to generate gene - level diversity , as suggested in chipman et al . ( 2014 ) . as duplication rather than splice variation has also been recently noted in the chelicerate ixodes scapularis ( brites et al . 2013 ) , it is likely that the alternative splicing of dscam , and perhaps other genes , has evolved independently in the pancrustacea ( chipman et al . 2014 ) .
) . these are distributed with 22 genes on the majority - strand ( α ) , and 15 on the minority - strand ( β ) . full details , along with start / stop codon and positional information can be found in
online , along with full mtdna sequence and primers used . of the 13 protein - coding genes , the typical metazoan start codon , “atn , ” is used by 12 , whereas
employs “acg . ” six genes , however , use an atypical incomplete stop codon . twelve genes overlap in coding sequence with another , and a total of 510 noncoding base pairs were noted , with 360 of these between the
) , followed by differential patterns of loss or mutation . this species’ mtdna gene arrangement generally closely resembles the unusual arrangement seen in its sister taxa and that of the spirostreptid
therefore seems generally conserved in spirobolid millipedes , and likely predates the divergence of the spirobolid and spirostreptid lineages .
as sister groups with maximal support . most local interrelationships are recovered as expected given previous knowledge ( e . g . ,
) and with firm bootstrap support . however , the location of the symphyla in particular in our analysis , as sister taxa to all other myriapoda , runs counter to many lines of evidence , which generally place symphyla as seen in
) using a range of models supports this topology . the placement of the pauropoda within the diplopoda is also interesting , given the extensive character loss which must be inferred if this is correct . difficulties in resolving deep phylogenetic relationships with mtdna data , both generally in the arthropoda (
) have been noted previously , and we suggest that deeper sampling in the myriapoda is a necessity if mtdna is to be used as a character for phylogenetic inference with confidence , given the high rates of change and reorganization seen in this clade .
insects have been the workhorses of genetics and developmental biology since that field began . we therefore know far more about how genes control development in insects than in any other clade in the arthropoda . with the publication of data from noninsect arthropods , our knowledge of the underpinnings of many developmental processes is maturing greatly ( e . g . ,
genome can also help this process . the sequences of many other key developmental genes and transcription factors are also found in the
genomic data set . high levels of recovery of several well - categorized gene families allow us to both confirm deep sequence recovery in our data set and clarify previously opaque areas of diplopod and arthropod molecular evolution .
) . after removal of all columns containing one or more gaps , a final , 57 amino acid alignment spanning the homeodomain was used as the basis of phylogenetic inference . bayesian phylogeny using the same sequences can be found in
online . numbers at base of nodes represent bootstrap percentages ( from 1 , 000 replicates ) . scale bar at top center represents substitutions per site at given unit distance . colored boxes denote individual hoxl gene families . note :
genes differ in sequence markedly from similar genes , and fall outside their known orthologs . tree rooted using
( underlined , red ) with those of known homology from other species , inferred by maximum - likelihood reconstruction . sequences downloaded from ncbi’s nr database have accession numbers as noted in next to taxa names , whereas others were sourced from the ensembl metazoa web resource (
) . after removal of all columns containing one or more gaps , a final , 68 amino acid alignment spanning the forkhead domain was used to infer phylogeny . bayesian phylogeny using the same sequences can be found in
online . numbers at base of nodes represent bootstrap percentages ( from 1 , 000 replicates ) . scale bar represents substitutions per site at given unit distance . tree rooted using
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the forkhead box ( fox ) gene class of transcription factors is responsible for mediating a broad range of cellular activity . known for its “winged helix” forkhead motif , derived from the helix - turn - helix class of genes to which they belong , these genes are easily recognizable and play key roles in metazoan growth and development (
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) , confirming further the sequencing depth of our genome . the identity of these was proven by phylogenetic inference , as can be seen in
) , confirming that losses in insects are specific to that clade . the only absentee from our data set is
the sox family of transcription factors was also targeted for detailed investigation . these hmg class genes play diverse roles in sex determination , growth , and development (
gene , confirming that this loss is specific to the centipede’s lineage rather than shared myriapod - wide . the only exception to the strong orthology with well - categorized sox clades comes in the form of a divergent hmg - box like sequence , found in both
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( underlined , red ) as inferred by maximum - likelihood reconstruction in mega . sequences of known homology were downloaded from ncbi’s nr database with accession numbers given on taxa labels or downloaded from the ensembl metazoa web resource (
) . all columns containing one or more gaps were removed and the resulting 80 amino acid alignment spanning the hmg domain was used to reconstruct phylogeny . bayesian phylogeny reconstructed with the same alignment can be found in the
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( underlined , red ) as inferred by maximum - likelihood reconstruction in mega . sequences were downloaded from ncbi’s nr database with accession numbers as noted in figure or sourced from the ensembl metazoa web resource (
) . after removal of all columns containing one or more gaps , a final , 88 amino acid alignment spanning the t - box domain was used to infer phylogeny . a bayesian phylogeny using this alignment is presented in
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tbx1 / 15 / 20 amino acid sequence . all sequences and alignments used can be found in
suggests that these genes could have duplicated early in the arthropod radiation and undergone extensive gene conversion in the interim , but the lack of evidence of paralogy could also mean that these loci are prone to independent duplication , for reasons unknown .
the recovery of developmentally important transcription factor genes in our data set is near - total . we show evidence of presence of in excess of 80 % of the expected transcription factor complements of a variety of key families , a considerable advance on presently extant data sets , and a figure which compares positively with that found in other genomes . these data will be vital for inferring the ancestral complements and functions of a variety of key genes across the arthropoda .
given the lifestyle of diplopods , the ability to sense and respond to a variety of environmental cues , and particularly the ability to assay the chemical composition of their habitat , is key to allowing them to find food and one another while avoiding predators and the worst environmental effects . the colonization of land by insects and myriapods occurred independently ( regier et al . 2010 ) , and thus it is interesting to consider whether chemosensory genes evolved before or after their diversification .
in s . maritima , no representatives of the odorant binding protein ( pelosi 1994 ) or chea / b families ( starostina et al . 2009 ) could be identified , leading to the conclusion that these genes are possible insect novelties ( chipman et al . 2014 ) . this is collaborated by our data set , where we were unable to note even putative homology to any sequence in our genome when we searched our data set using a number of genes from these families ( tblastn , e cutoff 1 ) . the chemosensory protein ( csp ) family ( pelosi et al . 2006 ) possesses two orthologs in our data set , as seen in s . maritima . these differ markedly in amino acid sequence from the centipede orthologs , however , which may reflect the differences in environments inhabited by these species .
our data set therefore corroborates the hypothesis that the odorant binding protein , chea / b , and or gene families are insect novelties . the csp , gr , and ir families are present in the rusty millipede , but differ greatly in sequence identity and complement number to that found in the centipede . this likely reflects the differences in environment and behavior exhibited by these species , but may also be a consequence of an increased tendency to duplicate genes for the creation of genetic diversity in s . maritima . increased sequencing efforts in the myriapoda will allow testing of these hypotheses further .
myriapods also represent possible sources of antifungal and antibacterial agents . these can be recognized by similarity to known genes in key domains . for instance ,
as with the known mycin sequences , these novel proteins possess clear similarity to the gamma - thionin ( pf00304 ) antifungal domains found in plants , but the differences in sequence outside the core domain mean that they may have novel characteristics . these and other novel bioagents may be of use and interest to a range of biochemical industries , given the often compromising niche inhabited by the millipede .
the resource detailed here represents a vital data set for beginning investigation into the evolution of a variety of traits in the diplopoda , myriapoda , and the arthropoda more generally . it is a draft genomic data set with deep coverage of the coding complement and high recovery of the total expected genome size . with a broader sampling of genomic diversity , our ability to infer the true origin of genes and phenotypes will allow us greater biological insight , and allow firm conclusions to be drawn as to the reasons for the success and diversity of the arthropod lineage . this data set also contains a variety of intriguing findings . millipedes were the first arthropods to emerge onto land , and possess a variety of unique adaptations which have contributed to their success over their 400 - myr old history . this genomic resource will allow us to investigate the diversity of millipede novelties , such as their defensive chemicals and fungicides , with much more vigor than previous studies have been able to accomplish .
we can also be more confident of assertions regarding myriapod genomics with the advent of this resource . for example , the extensive gene duplication seen in s . maritima is far less prevalent in this resource . although some duplication as a means to build genomic diversity seems to occur in this clade , it is of much more limited scope , and the scale of gene duplication seen in the centipede should therefore not necessarily be expected in other members of the myriapoda . our recovery of transcriptomic cassettes also allows a different angle to examine origin and loss of traits in arthropods from a molecular perspective—we can confirm , for instance , the presence of dmbx , foxj2 / 3 , and foxl1 in the arthropod common ancestor , and show firm evidence for the existence of tbx 4 / 5 and soxf in this lineage .
the advent of myriapod data sets will also allow a comprehensive revisiting of how extant gene complements were co - opted into the formation of new organs , particularly oxygen exchange systems , after terrestrialization in insects and myriapods ( e . g . , grillo et al . 2014 ; sánchez - higueras et al . 2014 ) . although genes involved in the development of respiratory organs , such as apterous ( e . g . , damen et al . 2002 , sequence ; supplementary file s1 , supplementary material online ) , are present in our data set , evidence remains to be gathered on any potential role of these genes in respiration in the myriapoda . this is because terrestrialization occurred independently in insects and myriapods , and we are cautious about inferring functional homology between genes in these species without expression data . further research using this genome as a resource will however offer a unique insight into how terrestrialization was accomplished independently in these speciose and successful metazoan clades .
as a developmental and genetic model , there is much that could be learnt from a millipede model species . segmentation , a subject of much interest in the insect developmental community , is presently still to be completely understood in the myriapoda , with janssen et al . ( 2004 ) suggesting that it may even be separately programmed dorsally and ventrally , at least in glomeris marginalia . millipedes may also be of developmental utility for understanding the original opening position of the genitalia in arthropods , given the peculiar cephalic location of this opening in this clade , and for understanding neurogenesis across the arthropoda ( e . g . , dove and stollewerk 2003 ) . millipedes also undergo periodomorphosis—adult to adult molts , with sexually mature and intercalary instars ( verhoeff 1923 ; sahli 1990 ) . understanding how this is regulated , in contrast with the final adult molts seen in other arthropods , is likely to benefit greatly from a genomic resource .
supplementary files s1–s4 are available at genome biology and evolution online ( urltoken ) .
the authors thank the afcd of the hk government for confirming identity of the millipede species . they also thank bgi for their help in sequencing . this work was supported by the lo kwee - seong biomedical research fund and lee hysan foundation to h - m . l . and j . h . l . h .
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to the surprise of the authors , their data led them to propose a clade that includes bats ( order chiroptera ) , carnivores such as cats and dogs ( order carnivora ) , horses and other odd-toed ungulates ( order perissodactyla ) and pangolins ( order pholidota ) as springing from a single evolutionary origin within the mammals .

the name pegasoferae was coined from the name of the mythological flying horse pegasus to refer to bats and horses , and the term ferae , encompassing carnivorans and pangolins .

according to this , the odd-toed ungulates ' closest living relatives are the carnivorans .

earlier theories of mammalian evolution would , for example , have aligned bats with the insectivores ( order eulipotyphla ) and horses with the even-toed ungulates ( order artiodactyla ) .

some subsequent molecular studies published shortly afterwards have failed to support it .

in particular , two recent studies , each combining genome-wide analyses of multiple taxa with testing of competing alternative phylogenetic hypotheses , concluded that pegasoferae is not a natural grouping .

pegasoferae

pegasoferae , an unexpected mammalian clade revealed by tracking ancient retroposon insertions . - pubmed - ncbi
pegasoferae is a proposed clade of mammals based on genomic research in molecular systematics by nishihara , hasegawa and okada ( 2006 ) .
this is a one - residue deletion in the second transmembrane helix of this shortwave imaging opsin considered under informative opsin indels . this is too phylogenetically narrow to illuminate pegasoferae .
this potentially informative retroposon insertion denoted int283 conflicts with pegasoferae topology . that is not a fatal flaw because a certain fraction of such events resolve anomalously via lineage - sorting after speciation .
pegasoferae is a proposed clade of mammals based on genomic research in molecular systematics by nishihara , hasegawa and okada ( 2006 ) . it includes bats , carnivorans , pangolins , and odd - toed ungulates .
nishihari et al located 4 retroposon insertion events that support pegasoferae . in the ensuing 2 - 3 years , it has become possible to reinvestigate these events with additional species utilizing bioinformatic methods on the 10 available laurasiathere genomes ( not all of which will completely cover a given event ) .
the one - residue indel appears cleanly restricted to pegasoferae . there are 5 laurasiatheres with it and 5 without it , which needs further buttressing by pcr ( adding more basal species has the effect of localizing the event farther back on the stem ) . the parental gene did not develop the indel in any bioinformatically accessible species .
it can always be asked whether this is the
same
l1ma9 in cetartiodactyls or merely a similar one from a separate insertion event of the active parental element . however pushing the putative event back to basal vicugna narrows the window for that . while this situation can be dismissed with an appeal to lineage - sorting ( importantly 4 events support pegasoferae versus this 1 conflict ) .
knowledge of the biological characteristics , physiology and behavior of the creature group pegasoferae . this group contains for instance bats , carnivorans ( the dog - and cat - like animals ) , and odd - toed ungulates such as horses and nosehorns . a higher skill increases taming and / or controlling chances , butchery yield , carcass cooking quality , and damage and defense for creatures in this group .
pegasoferae is a novel proposal using rare genomic events involving retroposon insertions to establish the phylogenetic ordering within laurasiatheres , grouping bats , perissodactyls and carnivores to the exclusion of the other hoofed mammalian group , artiodactyls . bats have been placed in many previous locations , notably in the euarchonta wing ( outgroup to primates ) . while that particular idea is clearly refuted by many lines of evidence , the proper placement of bats remains under discussion .
despite more intensive phylogenetic sampling , int391 continues to support pegasoferae as nishijimi et al originally stated . it should be noted that the mer - class retroposon , while not at issue here , exhibits the type of homoplasy that makes retroposons dicey as tree topology markers . introns are often susceptible to multiple insertions of similar retroposons as well as to complicated patterns of micro deletions that prevent their recognition even if they aren ' t fully deleted .
the last exon of rbp3 , like in many proteins , does not appear to be under a great deal of selective pressure . this surfaces in a certain amount of observed ' terminal wander ' as various mutations allow readthru of the original stop codon to the next one that occurs by chance downstream . here pegasoferae , relative to artiodactyls , all share a one bp indel within coding that causes an altogether novel amino acid sequence to terminate the protein .
this gene duplication might simply be restricted to this one genus ( or even this one species ) . alternatively it might have greater phylogenetic depth and be somewhat informative in resolving intra - bat taxonomy . this would require pcr of many additional bat species since no further genomic work is anticipated any time soon . it is not relevent to pegasoferae per se since bats are clearly monophyletic overall , even as the macro / micro distinction remains equivocal as a taxonomic character .
again , this presented a favorable annotation situation because the two flanking exons are well - conserved and the intonic distance is short at about 1500 bp . using new genomic data , the species density can be brought up 10 laurasiatheres , tiling traces in unassembled genomes as necessary . the conflict with pegasoferae holds up even sampling at this greater taxonomic sampling depth : an orthologous l1ma9 is also missing in microbat , shrew , hedgehog but present with correct orientation and correct fragment coordinates in vicugna , pig , and dolphin .
an example of int391 locus suggesting pegasoferae clade . ( a ) an electrophoretic profile of pcr products of locus int391 , in which l1 is present in horse , cat , and bat , but not other mammals . the larger size of the mouse pcr product is caused by species - specific insertions of another retrotransposon ( database position chr19 : 55196706–55197619 in mm7 ) . m , size markers ( øx174 / hincii digest ) . ( b ) an alignment of locus int391 sequences . thick and thin lines denote the l1 and the direct repeat sequences , respectively , that were generated during integration . the central region of the inserted l1 sequence is omitted .
the history here is quite complex when tetrapods outside laurasiatheres are included . ancestrally , the exon was clearly much shorter , terminating at consistent length just after module m4 ended in the rpb3 internal repeat structure from frog to marsupial divergence . in afrotheres , either the stop codon mutated or a frameshift occured , causing the protein to become longer . the new stop codon in hyrax represents the ancestral placental position . elephants retain a cryptic stop codon here but evolved an earlier one that is used today . this stop codon and some aspects of sequence was conserved in pegasoferae but not artiodactyls ( though by reinstituting the original trinucleotide ggg and former reading frame , residual ancestral sequence as well as the original distal stop codon can still be seen . shrew and hedgehog diverged by a different scheme and are unhelpful .
discover a faster , simpler path to publishing in a high - quality journal . plos one promises fair , rigorous peer review , broad scope , and wide readership – a perfect fit for your research every time .
molecular studies have reported divergence times of modern placental orders long before the cretaceous–tertiary boundary and far older than paleontological data . however , this discrepancy may not be real , but rather appear because of the violation of implicit assumptions in the estimation procedures , such as non - gradual change of evolutionary rate and failure to correct for convergent evolution .
new procedures for divergence - time estimation robust to abrupt changes in the rate of molecular evolution are described . we used a variant of the multidimensional vector space ( mvs ) procedure to take account of possible convergent evolution . numerical simulations of abrupt rate change and convergent evolution showed good performance of the new procedures in contrast to current methods . application to complete mitochondrial genomes identified marked rate accelerations and decelerations , which are not obtained with current methods . the root of placental mammals is estimated to be ∼18 million years more recent than when assuming a log brownian motion model . correcting the pairwise distances for convergent evolution using mvs lowers the age of the root about another 20 million years compared to using standard maximum likelihood tree branch lengths . these two procedures combined revise the root time of placental mammals from around 122 million years ago to close to 84 million years ago . as a result , the estimated distribution of molecular divergence times is broadly consistent with quantitative analysis of the north american fossil record and traditional morphological views .
by including the dual effects of abrupt rate change and directly accounting for convergent evolution at the molecular level , these estimates provide congruence between the molecular results , paleontological analyses and morphological expectations . the programs developed here are provided along with sample data that reproduce the results of this study and are especially applicable studies using genome - scale sequence lengths .
citation : kitazoe y , kishino h , waddell pj , nakajima n , okabayashi t , watabe t , et al . ( 2007 ) robust time estimation reconciles views of the antiquity of placental mammals . plos one 2 ( 4 ) : e384 . urltoken
copyright : © 2007 kitazoe et al . 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 .
funding : this work was supported by the technology agency ( jst ) and the japan society for the promotion of science ( jsps grant sr b - 16300086 to yk and hk ) . pjw acknowledges the jsps senior fellowship program and nih grant 5r01lm8626 for support .
despite great progress over the past decade , the evolutionary history of placental mammals remains controversial . while a consensus is emerging on the topology of the evolutionary tree [ 1 ] – [ 5 ] , although with occasional disagreement [ 6 ] – [ 8 ] , divergence times remain uncertain . the age of earlier nodes and in particular the root , remain especially uncertain in the absence of definitive placental fossils deeper into the cretaceous [ 4 ] – [ 6 ] , [ 9 ] . both paleontological and morphological studies suggest that the radiation of placental orders and super orders occurred close to the cretaceous–tertiary ( k–t ) boundary about 65 million years ago ( mya ) [ 9 ] , [ 10 ] .
in contrast , molecular studies have suggested markedly older origins for many superordinal groups and that some extant orders diversified before the k–t boundary . the root of living placental mammals has been reported to be in the range of 100–140 mya [ 4 ] , [ 11 ] – [ 15 ] even with application of rate - adjustment techniques [ 4 ] , [ 15 ] . the molecular consensus of an old root is becoming strong enough that it may become dogma without a close examination of the assumptions being made in such analyses . the true age of the orders and superorders has important implications for determining the overall paleoecology and biogeography of placental mammals during a period that included the breakup of continents and the extinction of terrestrial dinosaurs . reconciling the different paleontological and molecular divergence time estimates has important implications for basic methodologies that are central to evolutionary study .
the strength of molecular divergence time studies is their potential to draw information from very long aligned sequences of many species . it is widely assumed that a huge amount of sequence data and the approximate rate constancy of sequence evolution [ 16 ] make molecular estimates more reliable than those based solely on fossil data . such analyses on a genomic scale are generally anticipated to be decisive because they are expected to be free of stochastic noise [ 17 ] , [ 18 ] .
however , molecular studies acknowledge the problem of misspecification of the model of sequence change , which may result in seriously biased estimation . the relationships among placental orders do vary according to the data used and the taxa sampled [ 1 ] – [ 8 ] , [ 13 ] – [ 15 ] , [ 19 ] – [ 22 ] . most methods of phylogenetic inference rely strongly on probabilistic models of sequence evolution , and neither directly detect nor correct for convergent evolution [ 23 ] . when left uncorrected , such “homoplasy” may attract lineages and underestimate certain pairwise distances . this in turn distorts branch ( edge ) length estimates , which are of primary importance for divergence time estimation . while some methods will detect potential convergent evolution , for example hadamard conjugations and splitstree [ 24 ] , kitazoe et al . have developed multidimensional vector space ( mvs ) representation methods to both detect convergent evolution among estimated distances and to also correct for this bias [ 25 ] – [ 27 ] .
a further problem facing molecular dating is the evolutionary rate constancy , or lack of constancy , over long periods . since its proposal in 1965 [ 28 ] , the molecular clock hypothesis has been one of the most hotly debated subjects in evolutionary biology . it is now widely accepted that a molecular clock tends to hold well for closely related organisms , but breaks down with increasing evolutionary divergence . to adjust for the inevitable fluctuation of evolutionary rates , nonparametric [ 17 ] , [ 18 ] , local clock [ 29 ] , [ 30 ] , and hierarchical bayesian [ 31 ] methods have been developed . these methods are robust against stochastic fluctuations of evolutionary rates [ 31 ] – [ 33 ] . however , they may cause serious problems when pronounced transient changes of rate occur , because they overly smooth such rate changes . in this article , we show the magnitude of such problems using clear worked examples .
to improve the detection of , and robustness to , abrupt rate changes , we have developed a new procedure that minimizes the local variability of the inverse of the evolutionary rate . just as the effective size of fluctuating populations is represented by the harmonic mean over time , the mean evolutionary rate among lineages is expressed better by the harmonic mean . this approach is especially useful when branch lengths measured in the expected number of substitutions per site ( the products of rates and times ) are estimated accurately , and there are either rapid transient changes of rate ( hence large rate heterogeneity ) or a general bias towards a speed up or slow down in rates through time .
using this new procedure , an analysis of 69 mitochondrial protein sequences ( 3660 amino acid sites in total ) from placental mammals identified a rapid acceleration of evolutionary rate for the lineage directly leading to the common ancestor of supraprimates and an even more marked one for the lineage leading to laurasiatheria . this acceleration was followed closely by a strong deceleration , which persisted in nearly all lineages of laurasiatheria . in contrast , almost all lineages of afrotheria and xenarthra seem to have retained rates similar to that of the root . this view is in marked contrast to current rate - change penalty functions . the robustness of the new procedure is assessed using simulations that show the types of change that most concern biologists ; speedups or slowdowns through time , transient rate changes , and rate changes that are do not follow a normal or transformed normal distribution , as well as stochastic variation . a revised estimate of the origin of placental mammals is as young as 84 mya , which is much more recent than current estimates using molecular data . the inferred age of deeper splits in the placental tree are compared with the rate of occurrence of new species from the north american fossil record . these two sources of data are far more congruent than is suggested by using current , possibly strongly misleading , dating methods .
mitochondrial protein sequences are used widely in phylogenetic studies and have been particularly popular in studying placental mammals . a desirable feature of these data is relatively long sequences , good taxon sampling and very little missing data . following alignment , we retained 3660 amino acid sites present in all of 62 placental mammals plus seven outgroup taxa ( table s1 gives the accession numbers of protein sequences ) . in contrast , large nuclear protein datasets of mammals with diverse taxon sampling [ e . g . 3 ] , [ 4 ] show a large proportion of missing data . such missing heterogeneous data may lead to complex systematic errors in distance estimation [ 34 ] . this is particularly relevant since mvs analyses work best with low stochastic noise ( hence relatively long sequences ) , diverse taxon sampling with a considerable proportion of taxa showing minimal convergent evolution , and minimal bias of the input distances from sources such as missing data .
we adopted a standard two - step procedure to estimate divergence times . the first step is to estimate the phylogenetic tree with unconstrained branch lengths in units of expected numbers of substitutions per site . given the problems with convergent evolution in mitochondrial data [ 4 ] , [ 5 ] , [ 22 ] , [ 25 ] , we used the mvs procedure to correct the input distances for convergent evolution . a variant of the core set approach [ 27 ] to mvs was used in this instance ( materials and methods ) . fixing the topology to the tree obtained by mvs , which is very similar to trees obtained by previous authors using varied data sets [ 2 ] – [ 5 ] , branch lengths were reestimated using maximum likelihood ( ml ) in the program paml [ 35 ] . we used the jtt [ 36 ] + gamma model with α = 0 . 5 for these analyses . the second step uses the tree and its branch lengths , some fossil constrained nodes , and a penalty or cost function to dampen rate changes , to infer divergence times of all nodes and evolutionary rates along all branches .
three cost functions , f add , f log , and f ir , were applied to the mvs and ml trees . these functions penalize the fluctuation of rates , and do this on either a linear scale ( here called the add function ) , on the log rate ( log ) , or on the inverse rates ( ir ) , respectively ( see equations ( 1 ) , ( 2 ) , and ( 4 ) in materials and methods ) . the tree estimated by mvs and f add is denoted mvs - f add and so on ( while ml - f add indicates use of ml branch lengths ) .
to calibrate these trees , we used eight fossil constraints , all taken from previous studies ( see figure 1 caption ) [ 4 ] , [ 15 ] . the divergence times were estimated by then minimizing each cost function subject to these constraints ( see materials and methods ) . the ci was calculated using a likelihood interpretation ( see materials and methods ) of the cost function residual analogous to the method used in multidivtime [ 31 ] . this captures the error caused by deviations of the tree ' s branch lengths away from their expected values and includes the unpredictability of rate changes , which might mimic brownian motion , for example . it will also incorporate variability arising from ancestral polymorphism . polymorphism will cause fluctuations of the edge lengths of the tree when the analysis has multiple nodes constrained by fossil or other data [ c . f . 13 ] . generally , the time at the most recent common ancestor of sequences from different species is older than the time at speciation . the extent of this difference varies among internal nodes due to both the stochastic nature of the coalescent and factors governing its expected magnitude , such as population size . the cost function does not take account of this bias , but the ci does include the variance arising from ancestral polymorphism at internal nodes .
a and b show the mvs - f ir and ml - f log trees , respectively . the numbers 1–61 denote the ancestral nodes . the red numbers 1–8 indicate the internal nodes with fossil constraints which are as follows : 1 , 49–61 ; 2 , 52–58 ; 3 , 45–63 ; 4 , 43–60 ; 5 , < 63 ; 6 , > 12 ; 7 , 36–55 ; 8 , 54–65 ( all in mya ) [ see 4 ] , [ 15 ] . the colors of the branches denote inferred evolutionary rates ( in units of ×10 −9 / site per year ) as follows : black , < 0 . 2 ; dark blue , 0 . 2–0 . 3 ; light blue , 0 . 3–0 . 4 ; green , 0 . 4–0 . 5 ; brown , 0 . 5–0 . 6 ; yellow , 0 . 6–0 . 7 ; and red , > 0 . 7 .
the mvs - f ir analysis ( figure 1a ) estimated the age of the root at 84 . 2 mya with a 95 % confidence interval ( ci ) of 80 . 7–88 . 4 mya ( table 1 ) . this time is much more recent than the result of the ml - f l analysis ( figure 1b ) , i . e . , 122 . 2 ( 95 % ci , 112 . 2–144 . 7 ) mya . note that , ml - f l is giving results consistent with that reported in earlier studies [ 1 ] – [ 5 ] , [ 12 ] – [ 15 ] , indicating that the younger root for placentals returned using the function f ir ( see below ) is due to the method and not the data . the difference between the f log and the f ' log function in table 1 is explained in materials and methods . it is the f log method that most closely approximates the brownian motion assumed by multidivtime .
figure 1 shows the estimated ancestral rates across the tree , while figure 2 represents the inferred evolutionary rates going from the root to a terminal . the single instance of root to fin whale is shown in figure 2 , while figure s1 traces the evolutionary rates inferred by different methods along seven representative lineages . the mvs - f ir tree identified an abrupt acceleration of evolutionary rate near the common ancestor of both supraprimates and laurasiatheria , then a very strong acceleration in just the ancestral lineage of laurasiatheria ( figures 1a and 2a ) . this acceleration was followed closely by a strong deceleration along nearly all lineages of laurasiatheria . in contrast , almost all lineages of afrotheria and xenarthra have retained rates close to that of the root . there are also sporadic later accelerations , for example that among hedgehogs and moon rat ( figure 1a ) .
figures a and b , respectively , trace the estimated rates along edges on analyses using mvs and ml branch lengths . the root time of of the f add analysis in figure a and b was set at a large value ( 400 mya ) because the numerical calculation continues towards an infinite root time .
the cost function f ir detected an acceleration–deceleration pattern near the base of laurasiatheria using both the mvs and ml branch lengths ( the red lines of figure 2 ) , whereas the function f log showed a far more flat prediction of generally lower evolutionary rates that led to older root times ( the green lines of figure 2 ) . even more extreme , the cost function f add inferred gradually decreasing rates in all deep branches of the mvs and ml trees ( the blue lines of figure 2 ) , and the root time tended to infinity . table 1 summarizes the root time values estimated by various models ( all the node divergence times from these models are listed in table s2 ) .
table 1 also compares the inferred times with those obtained from r8s , a popular program for estimating absolute rates [ 17 ] , [ 18 ] . this program has two options of for its cost functions , f add and f′ log ( equation ( 3 ) in materials and methods ) . it also takes account of stochastic noise caused by the finite length of the sequences used to derive the branch lengths using a penalized - likelihood approach . the fitting to the estimated branch lengths is expressed by the log likelihood , while the weight for the cost function is estimated using cross validation . in our mitochondrial dataset , with its long sequences , the root time values changed little due to the cross validation effect ( the reason is explained in materials and methods ) . further , root divergence time estimates returned by r8s do not show confidence intervals in table 1 , since r8s only assesses the sampling variance of node times due to stochastic errors in branch lengths due to finite sequence length ( it does this via the bootstrap ) and does not include the effect of stochastic variation of evolutionary rate . this source of error remains as sequence lengths go to infinity , and will be dominant with long sequences .
figure 3 represents the chronological distribution of the internal node density for the mvs - f ir tree ( figure 1a ) compared with the rate of new species appearing in the well - studied north american fossil record . to avoid potential bias of divergence times in the molecular tree due to species sampling , the scaling constant was determined by a least - squares fit to the fossil data in the period 85–50 mya .
the blue squares show the rate of the appearance of new species based on the fossil record [ adapted from 3 ] . at present , such quantitative data are limited to the well - studied north american record . the red circles represent the chronological distribution of node density ( or splits ) on the mvs - f ir tree ( figure 1a ) . the green triangles represent split frequency on the ml - f log tree ( figure 1b ) . the node density is given by the number of nodes in an 8 myr sliding window . a scaling constant for the molecular frequencies was calculated via a least - squares fit to the fossil data in the period 85–50 mya .
the mvs - f ir analysis reconciles molecular with fossil data in two ways . first , the chronological distribution of the internal node density is clearly largely consistent with the rate of appearance of novel fossil species . this is despite the fact that the paleontological data assessed are limited to the well - studied north american record [ 10 ] . both the mvs - f ir tree and the fossil record suggest accelerating taxonomic diversity near the k–t boundary , rather than a longer slower buildup [ 2 ] – [ 5 ] , [ 12 ] – [ 15 ] . use of the less robust f log criterion seems to suggest a prolonged increase in diversity that agrees far less with the quantitative fossil record . this congruence with fossils does not automatically show that the combination of mvs and f ir is the best way to analyze this data , but it does reframe the discussion of the relationship between the fossil and molecular times into one where the molecular dates are being looked at far more critically .
second , and more indicatively , the cost function f ir resolves incongruence among the fossil constraints / inferred divergence times in different parts of the molecular tree . the best fossil constraints in laurasiatheria suggest much older times than constraints in other parts of the tree [ 4 ] , [ 37 ] . the mvs - f ir , mvs - f log , and mvs - f add trees with all constraints give the root as 84 . 2 , 105 . 0 , and ∞ mya , respectively ( table 1 ) . removing the whale–hippo constraint gives 82 . 4 , ∞ , and 91 . 2 mya ; removing the horse–rhino constraint gives , 82 . 8 , 105 . 0 , and ∞ mya ; and removing both sets of constraints gave 82 . 9 , 87 . 2 , and ∞ mya . thus , in this case only the f ir tree is insensitive to “constraint sampling” .
finally , there is a good fossil calibration for tarsier [ 4 ] that was withheld because it is not used in both references 4 and 15 . it suggests that human and tarsier split around 50–60 mya and the molecular trees suggest the human - loris split was not much earlier ( probably < 5 myr earlier ) . despite our use of whale–hippo and horse–rhino calibrations , the mvs - f ir tree ( figure 1 ) gives a human - loris split close to this age , in contrast to earlier studies [ 4 ] , [ 15 ] , [ 33 ] .
we highlight two distinct properties of the new function f ir with the help of evolutionary simulations and worked examples . the first property is its ability to detect a transient acceleration of evolutionary rate . such an effect might be caused by a burst of positive selection and / or a bottleneck in population size . the second is to assess the effects of both stochastic fluctuations and systematic bias on the robustness of estimated times . here , we model bias in the form of either a general slowdown or a general acceleration of evolutionary rate across the whole tree .
we first modeled a strong instantaneous acceleration as an analogue to what is inferred by f ir to have occurred ancestral branch leading to laurasiatheria . we simulated a 32 - taxon symmetric tree in which a molecular clock holds except for an abrupt elevation ( by a factor of 10 ) of evolutionary rate along a short internal branch ( the red line in figure 4a ) . this example is useful for demonstrating the efficacy of the f ir function and something similar appears on both the mvs - f ir and ml - f ir mitochondrial trees . the times at the internal nodes were set to 48 , 56 , 64 , and 72 mya , and the root time was set to 80 mya . on this weighted tree , the cost functions were minimized with two constrained node times , which corresponded to two fossil calibration points . only the f ir function accurately estimated the true divergence times and appeared robust against the abrupt change ( figure 4b ) . in contrast , the functions f log and f add inferred gradual rate changes ( figure 4e ) , which are erroneous and lead to overestimation of the root time along with that of many other nodes ( figures 4c and 4d ) . table 2 summarizes the root time values inferred by various models . table 2 includes the result of r8s with the cross - validation method .
a worked example of the effect of a transient elevation of evolutionary rate upon estimated divergence times .
in this worked example using a symmetric 32 - taxon tree ( figure a ) , a global molecular clock holds , except for a short - term increase in evolutionary rate along one branch ( the red line in figure a ) . the true root time was set to 80 mya , and the times at the internal nodes are 48 , 56 , 64 , and 72 mya . the deep internal branch ( the red line in figure a ) is given an evolutionary rate ten times that of the remaining edges . the various cost functions were minimized subject to two calibrated nodes ( the red numbers 1 and 2 in figure a ) , using the exact branch lengths of this example as input data . the cost functions f ir , f log , and f add inferred the weighted trees of figures b , c and d , respectively . figures e–g show the trace of evolutionary rates along the lineages from the root to taxa numbers 5 , 9 , and 25 of figure a , respectively . the inferred age of the root for each cost function is shown with an arrow . the function f ir recovered the original pattern of rate change , whereas the other two functions inferred far more gradual changes , which resulted in a substantial overestimation of the root time .
we next simulated stochastic rate fluctuations by themselves , plus either a prevailing slowing down or acceleration of rates through time . such simulations are distinct from a brownian - type process , and are used to gauge the general robustness of the functions . to impose rate fluctuations on the same basic tree as figure 4a , but without the abrupt rate change , the age of the root was set to 100 mya . we assumed infinite length of sequences and that the branch lengths are known without uncertainty . next a branch was randomly selected proportional to its duration in time and all its descendant branch lengths were multiplied by a factor ( chosen randomly ) from a uniform distribution of range 0 . 5–1 . 5 . a total of 25 such rate changes were placed on the tree to give the final branch lengths for that tree . these branch lengths were used as the input data and the various cost functions were minimized with on calibrated internal node , and then determined all node times . we repeated this procedure 600 times to obtain the mean and standard error of the root time . we also simulated the two other cases . the only difference was the range of the uniform distribution used . using a range of 1 . 0–1 . 75 the model is biased toward rate acceleration through time , while using and 0 . 25–1 induces a rate slow down through time ( a probable situation in the placentals generally ) . as table 3 shows , the function f ir gave reasonable estimates with a bias towards deceleration , whereas f add gave an infinite root time in 139 of 600 samples . these undefined root - time values were set arbitrarily to 200 mya before calculating the mean and standard error .
the mvs model was shown previously to recover the correct tree in a simulation with two strongly convergent lineages [ 26 ] that were grouped erroneously by standard methods ( including the neighbor joining ( nj ) [ 38 ] and ml [ 35 ] methods ) . here , we examined a different question ; this is how well the mvs method recovers the true branch lengths , which are of primary importance when estimating divergence times . we simulated the evolution of a sequence of 10 4 amino acid sites , following the tree depicted in figure 5 . the model of amino acid substitutions used was the jtt [ 36 ] . convergent evolution was then imposed on this data . this was done by sharing parts of the sequences among three ingroup clades plus the outgroup . the red lines in figure 5 indicate which lineages shared sequence and were therefore subject to a form of convergent evolution .
amino acid substitutions were evolved on the shown weighted tree using the jtt model [ 36 ] of amino acid substitutions . after this , 30 % of sites were swapped between the four lineages indicated by the red lines .
pairwise distances were then estimated from the terminal sequences obtained above using the same jtt model . a modified mvs core - set procedure [ 27 ] was able to recognize that the tree consisted of three groups of eight sequences which had additive distances within each group and the outgroup . the mvs procedure then converted the distances within the three ingroups into three sets of perfectly additive distances and sequentially combined them to form a single core set ( materials and methods ) . the final mvs tree was obtained by modifying the distances between this single ingroup core set and the outgroup following the rules described in materials and methods .
the branch lengths recovered by the mvs model reproduced the true values accurately ( figure 6 ) . the distribution around the diagonal line in figure 6 represents the stochastic fluctuation of the estimated distances ; that is , the magnitude of this fluctuation did not change after the simulation was rerun without convergent evolution . in contrast , the nj and ml trees returned branch lengths that were affected clearly by convergent evolution . the worst affected branch lengths were ancestral to the groups showing convergent evolution ( underestimated ) or else were leading to the sister groups of the groups affected by convergent evolution ( overestimated ) .
the tree topology inferred by these methods was identical to the tree that generated the data , so the estimated branch lengths are plotted against their true values . the blue numbers show the branch index , as used on figure 5 , of outliers . note , all the outliers are internal branches ancestral to the four lineages undergoing convergent evolution or are ancestral to the sister group to these lineages . branch lengths ancestral to the groups undergoing convergent evolution are underestimated by the ml and nj methods , whereas those ancestral to their sister taxa are overestimated .
we begin the discussion by examining why the current cost functions , f log and f add , overestimated the age of the root in the worked example with strong rate heterogeneity in the form of a short term highly elevated evolutionary rate . it is also important to examine the profile of the cost function around their minimal values for the age of the root . the functions f log and f add showed asymmetric behavior around the estimated root time , even in worked examples with a perfect molecular clock ( data not shown ) , whereas the f ir profile was symmetric and parabolic in shape . the asymmetry seen with f log and f add increased in response to a general bias towards deceleration of rates through time ( figure 7a ) . in particular , the function f add showed a monotonic decrease with respect to increasing age of the root , that is , its estimate tended to infinity . thus , asymmetric behavior of a cost function seems to be a symptom of unstable estimation of the age of the root . a similar strong asymmetry appeared in the profile of the current cost function with respect to the age of the root of the mitochondrial tree of placental mammals ( figure 7b ) , irrespective of whether mvs or ml branch lengths were used . comparing figures 7a and 7b suggests that bias due to decelerating evolutionary rates occurred in the evolutionary history of placental mammals , and is impacting the ability of current methods to estimate the correct age of the root .
this figure shows the profile of the cost function with respect to the age of the root estimated by three different cost functions . because the root age estimated by function f add was going to infinity , it was set to 200 mya for illustrative purposes . figure a is a single example from a tree simulated under the scenario of random auto - correlated changes of rate moving towards the tips , strongly biased towards a deceleration of evolutionary rates as time progresses ( from the set of simulations used for table 3 ) . the true age of the root was 100 mya . figure b shows the results using the mvs tree derived from the mitochondrial sequences of placental mammals . the dotted line indicates the 95 % confidence interval of the estimated age of the root using the sum - of - squares approach described in materials and methods .
other approaches to divergence time estimation are under active development . for example , drummond et al . [ 39 ] recently developed a set of bayesian procedures to jointly estimate divergence times , evolutionary rates , and the tree topology . the uncorrelated variable rate model they use , which assumes independence of evolutionary rates between branches , may also be able to identify instantaneous accelerations . however , the magnitude of rate acceleration may be underestimated because hierarchical bayes estimates can generally be regarded as shrinkage estimators [ 40 ] . an important future direction is to assess the performance of the various estimators in simulations where rate variability is a mixture of an autocorrelated process plus rare , but strong , instantaneous accelerations .
our refined approaches resolve apparent contradictions between the quantitative molecular and paleontological data of placental mammals . given such agreement , there is no need for ancillary hypotheses such as the long fuse model [ 2 ] , [ 5 ] , [ 9 ] , [ 12 ] – [ 15 ] , [ 41 ] to explain the lack of any positively identified fossils of modern placental mammals prior to about 75 million years ago . in addition , dates of markedly less than 100 mya for the root of placental mammals also bring into question earlier hypotheses that traditional continental drift models explain the geographic distribution of the four major groups of placental mammals [ 2 ] – [ 5 ] , [ 13 ] , [ 42 ] . we anticipate that the f ir cost function developed in this paper will provide an improved methodology for a wide range of molecular studies because its robustness to rapid fluctuations of rate is essential to understanding events such as adaptive evolution . for example , because acquisition of new molecular functions can be achieved in a few million years after gene duplications [ 40 ] , the duration of an inflated evolutionary rate may be surprisingly short , and , correspondingly , difficult to detect . we hope our new methods will be beneficial in such situations and will lead to a richer understanding of molecular evolution .
because the branch lengths of a phylogenetic tree are estimated from the data as the product of the evolutionary rate of a branch and its time duration , these two factors cannot be directly estimated separately . it has become standard to use loose constraints to accommodate uncertainty and it is often wise to exclude constraints if there is no firm basis for them . information on the age of some internal nodes may be fairly directly available from the fossil record . an example of this is the horse - rhino split [ 13 ] . if the molecular clock [ 16 ] governs the process of molecular evolution well , we can accurately estimate the times at other nodes given just a single reliable calibration point . however , the assumption of the molecular clock is often rejected by the fit of a clock - like tree to the data .
the most widely used assumption in order to model the evolutionary rate changes away form being constant ( or away from a clock ) is to introduce a stochastic process . for example , sanderson
that rate changes follow a random walk with independent increments randomly drawn from a normal distribution . likewise , thorne et al .
uses rates at the nodes , instead of average rates along branches , as the free parameters ) . this can be interpreted as an assumption that the log rate undergoes brownian motion . that is , independent increments of normal random variables , whose variances are proportional to the average time duration between the pair of branches ( that is , the average length in time of the two branches ) . recently , sanderson implemented the unweighted cost function
however , the estimated rates based on any of the above cost functions may overly smooth the change of evolutionary rates when a pronounced transient change of rate has occurred . in turn , biased estimates of evolutionary rates will lead to biased estimates of divergence times . here we propose a new cost function , which penalizes the local rate deviation from the harmonic mean . for simplicity , we ignore the stochastic variance of branch length
, which goes to zero with increasingly long sequences . denote the branch length and inverse rate of the
because f ir places a smaller penalty on abrupt rate changes than do previous models , it can confine an abrupt change close to where it occurred on the tree . in contrast , f add and f log a priori put stress upon the smoothness of evolutionary rate change from one branch to the next , and this may propagate the effect of an abrupt rate change over successive branches . in this article , the variable ( t n ) ( the divergence times ) were estimated by minimizing the cost functions ( equations 1–4 ) subject to the ( fossil ) constraints without introducing other modifiers such as cross validation .
at first glance , it seems most intuitive to use a penalty such as ( rate 1 −rate 2 ) 2 ; this has been the implicit assumption until now [ 31 ] and is the basis of published programs such as r8s and multidivtime [ 17 ] , [ 18 ] , [ 31 ] . it rests on the implicit expectation that any reasonable type of smoothing of changes in rate will give a similar answer . however , a simple linear form for the difference in rates is misleading , and this is illustrated by considering the general problem of estimating velocity ( rate ) given distance ( in our case branch length ) . going from point a to b at velocity v 1 , then back at velocity v 2 , the average velocity ( v av ) is equal not to the standard arithmetic mean , but to the harmonic mean , 1 / v av = ( 1 / v 1 + 1 / v 2 ) / 2 . further , going from point ( node ) a to b at velocity ( rate ) v 1 , and from point b to c at velocity v 2 , then the distances traveled ( d ab and d bc ) need no longer be equal so we must use weights . specifically , v av = ( d ab + d bc ) / ( t 1 + t 2 ) , where t 1 = d ab / v 1 and t 2 = d bc / v 2 . as a result , 1 / v av = ( d ab / v 1 + d bc / v 2 ) / ( d ab + d bc ) . this is the same form as the average quantity a n used to obtain f ir ( equation 4 ) . the use of the harmonic mean becomes important when v 1 differs greatly from v 2 .
the bayesian approach estimates the divergence times and evolutionary rates in the form of a posterior distribution , which is summarized approximately as p ( b | r , t ) exp ( −λf ) . here b , r , and t are the vectors of the estimated branch lengths , evolutionary rates , and divergence times , respectively , while f is the cost function . the value of λ expresses the weight for the penalty , and is called a hyperparameter . introducing the distribution for the hyperparameter is done via a so - called hyper - prior ; the hyperparameter is then estimated concurrently with the posterior distribution . the penalized likelihood approach maximizes log p ( b | r , t ) − λf . the weight for the penalty λ is estimated using cross - validation . when the sequences are long enough , for example concatenated mitochondrial protein sequences , it is often safe to assume that the branch lengths are estimated accurately , that is with minimal stochastic error ( but not necessarily without systematic error , something we address in this paper using mvs ) . accordingly , it is assumed that the products of rates and times are known exactly . thus in a situation of long , or very long sequences ( e . g . genomic alignments ) , the results returned by using just the penalty function will converge to those returned by either a bayesian or a penalized likelihood approach with the same type of cost function .
in the mvs method , the additivity of evolutionary distances is converted using the expected orthogonality among branch vectors in a multidimensional euclidean space [ 25 ] . if the estimated pairwise distances do not satisfy additivity , mvs provides an index to measure deviations from orthogonality without specifying a tree structure . this index enables one to diagnose whether a distance matrix is compatible with a tree and to modify the biased distances until they satisfy orthogonality , that is , with a zero value for the index . here , a revised core - set approach , that is , an extension of [ 27 ] , is applied to correct the observed pairwise distances for convergent evolution .
the first step of this approach is to divide the set of taxa into subgroups and to correct the distances between pairs in each subgroup . we decompose the taxa based on partitions with both strong biological support and high bootstrap support . the deviation from additivity of pairwise distances is much smaller within each subgroup than across the whole distance matrix . if an anomalously large deviation is observed associated with a single taxon within a subgroup , that taxon is removed temporarily from the analysis . when the only deviations between distances within a subset of taxa are judged to be due to stochastic noise , the pairwise distances are modified by solving the equation of motion ( a method using a many body kinetic equation in physics ) , which uses the index of the deviation from additivity as the potential energy [ 25 ] , [ 26 ] . the modified distance matrix within a subgroup is interpreted as the true additive distance matrix ( core set ) achieved by minimal modification of the original distances . generally , the modified distances are very close to the distances estimated by the nj method for just these taxa . then , the excluded taxa are deposited into this core set tree by solving the equation of motion with the same potential function . assuming that convergent evolution and long - branch attraction are the main source of deviation from additivity , we allow for only positive corrections of the biased distances between the core set and excluded taxa ( that is , distances can only get bigger ) .