Mammals genome - platypus

1) This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic

lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. 

2) Analysis of the

first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins

have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying

eggs; and immune gene family expansions are directly related to platypus biology. 

. 

3. Expansions of protein, non-protein-coding

RNA and microRNA families, as well as repeat elements, are identified. 

4) The platypus genome, as well as the animal, is an amalgam of

ancestral reptilian and derived mammalian characteristics. The

platypus karyotype comprises 52 chromosomes in both sexes14,15,

with a few large and many small chromosomes, reminiscent of reptilian

macro- and microchromosomes. Platypuses have multiple sex

chromosomes with some homology to the bird Z chromosome16.

Males have five X and five Y chromosomes, which form a chain at

meiosis and segregate into 5X and 5Y sperm17,18. 

5) Non-protein-coding genes

In general, the platypus genome contains fewer computationally predicted

non-protein-coding (nc)RNAs (1,220 cases excluded high

repetitive small nucleolar RNA (snoRNA) copies; see below) than

do other mammalian species (for example, human with 4,421 Rfam

hits), similar to observations in chicken19 (655 Rfam-based ncRNAs).

This is probably because of the extensive retrotransposition of

ncRNAs in therian mammals and the apparent lack of L1-mediated

retrotransposition in chicken and platypus. The exception to this is

the platypus family of snoRNAs, which is markedly expanded

(,2,000 matches to the Rfam covariant models) compared to that

for therian mammals (,200). snoRNAs are involved in RNA modifications,

in particular of ribosomal RNA, and are often located in

introns of protein-coding genes22. 

6. Our investigations revealed a

novel short-interspersed-element (SINE)-like, snoRNA-related

retrotransposon—which we have labelled snoRTEs—that has duplicated

in platypus to ,40,000 full-length or truncated copies. It is

retrotransposed by means of retrotransposon-like non-LTR (long

terminal repeat) transposable elements (RTE) as opposed to the

L1-mediated transposition mechanism in therians23. 

7 We constructed

a complementary DNA library of small, ncRNAs and identified 371

consensus sequences of small RNAs that included 166 snoRNAs23

(Supplementary Table 3). Ninety-nine of these cloned snoRNAs

are found in paralogous families, and 21 of them belong to the

snoRTE class. The presence of both the structural requirements

known to be important in snoRNA function24 and evidence of their

expression are consistent with these snoRTE elements being functional

in the platypus. Similar to other unrelated ncRNAs that have

proliferated in therian mammals (for example, 7SL RNA-derived

primate Alu elements, tRNA-derived rodent identifier (ID) elements),

this recent SINE-like expansion is probably due to chance

events. However, given the RNA modification activity of snoRNAs,

and our increasing awareness of the cellular importance of RNA

molecules, it might be that some of the retrotranspositionally duplicated

RNAs were exapted into new functions in this species.

Other small RNAs. Overall, we found commonalities with small

RNA (sRNA) pathways of other mammals, but also features that

are unique to monotremes. Components of the RNA interference

machinery are conserved in platypus, including elements of biogenesis

pathways (Dicer and Drosha) and RNA-interference effector

complexes (argonaute proteins; Supplementary Table 4). Of

20,924,799 platypus and echidna sRNA reads derived from liver,

kidney, brain, lung, heart and testis, 67% could be assigned to

known microRNA (miRNA) families. Established patterns of

miRNA expression were generally recapitulated in monotremes.

To determine the conservation patterns of miRNAs in platypus, we

identified platypus miRNAs sharing at least 16-nucleotide identity with

miRNAs in eutherian mammals (mouse/human) and chicken.

Although most conserved miRNAs were identified across these vertebrate

lineages (137 miRNAs), 10 miRNAs were shared only with eutherians

(mouse/human) and 4 only with chicken (Fig. 2a). miRNAs can

be classified into families based on identity of the functional ‘seed’

region at position 2–8 of the mature miRNA strand. We identified

miRNA families that were shared between platypus and eutherians

but not chicken (40 families), or between platypus and chicken but

not eutherians (8 families), suggesting that for some miRNAs only

the seed region may have been selectively conserved (Fig. 2a).

Conserved miRNAs tended to be more robustly expressed in the

platypus tissues analysed than lineage-restricted miRNAs (Fig. 2b).

To identify miRNAs unique to monotremes we used a heuristic

search that identifies miRNA candidates in deep-sequencing data

sets25. This method predicted 183 novel miRNAs in platypus and

echidna (Fig. 2a). Notably, 92 of these lay in 9 large clusters, on

platypus chromosome X1 and contigs 1754, 7160, 7359, 8388,

11344, 22847, 198872 and 191065. Physical mapping confirmed that

at least five of these contigs are linked to the long arm of chromosome

X1 (ref. 25). These abundantly expressed clusters were sequenced

almost exclusively from platypus and echidna testis (Fig. 2b). The

expansion of this unique miRNA class and its expression domain

suggest possible roles in monotreme reproductive biology25.

Piwi-interacting RNAs (piRNAs) associate with a germlineexpressed

clade of argonaute proteins, known as Piwis26, and have

a role in transposon silencing and genome methylation26. Monotreme

piRNAs bear strong structural similarity to those in eutherians.

They are,29 nucleotides in length and arise from large testis-specific

genomic clusters with distinct genomic strand asymmetry, often with

a typical ‘bidirectional’ organization. We identified 50 major platypus

piRNA clusters as well as numerous smaller clusters25. In contrast

to piRNAs in mouse, platypus piRNAs are repeat-rich and bear

strong signatures of active transposon defence.

8) Gene evolution

Overall this resulted in 18,527

protein-coding genes being predicted from the current platypus

assembly. 

As expected, the majority of platypus genes (82%; 15,312 out of

18,596) have orthologues in these five other amniotes (Supplementary

Table 5). The remaining ‘orphan’ genes are expected to primarily

reflect rapidly evolving genes, for which no other homologues are

discernible, erroneous predictions, and true lineage-specific genes

that have been lost in each of the other five species under consideration.

Simple 1:1 orthologues, which have been conserved without

duplication, deletion or non-functionalization across the five mammalian

species, were greatly enriched in housekeeping functions,

such as metabolism, DNA replication and mRNA splicing 

8. Chemoreception. The semi-aquatic platypus was expected to sense

its terrestrial, but not aquatic, environment by detecting airborne

odorants using olfactory receptors and vomeronasal receptors (types

1 and 2: V1Rs, V2Rs). Nevertheless large numbers of odorant receptor,

V1R and V2R homologues (approximately 700, 950 and 80,

respectively) are apparent in the platypus genome assembly, although

for each family only a minority lack frame disruptions (approximately

333, 270 and 15, respectively)34. The large expansion

of the platypus V1R gene family might reflect sensory adaptations

for pheromonal communication or, more generally, for the detection

of water-soluble, non-volatile odorants, during underwater

foraging.

The platypus odorant receptor gene repertoire is roughly one-half

as large as those in other mammals37. Nevertheless, platypus odorant

receptors fall into class, family and subfamily structures that are well

represented from across the mammals, with a few notable exceptions

such as family 14 (Fig. 3a). Together with the finding that lizard

contains only ,200 odorant receptor genes and pseudogenes, this

indicates that the platypus olfactory repertoire is, as expected, more

akin to other mammals than it is to sauropsids.

10. Eggs. Fertilization in the platypus exhibits both sauropsid and therian

characteristics. Platypus ova are small (4mm diameter) relative

to comparably sized reptiles and birds, and eggs hatch at an early

stage of development so that most growth of the embryo and infant is

dependent on lactation, as in marsupials. Like all mammals and

many other amniotes, when fertilization occurs the ovum is invested

with a zona pellucida. The platypus genome encodes each of the four

proteins of the human zona pellucida38, as well as two ZPAX genes

(Table 1) that previously were observed only in birds, amphibians

and fish. The aspartyl-protease nothepsin is present in platypus, but

has been lost from marsupial and eutherian genomes (Table 1). In

zebrafish, this gene is specifically expressed in the liver of females

under the action of oestrogens, and accumulates in the ovary39.

These are the same characteristics as of the vitellogenins, indicating

that nothepsin may be involved in processing vitellogenin or other

egg-yolk proteins. We find that platypus has retained a single vitellogenin

gene and pseudogene, whereas sauropsids such as chicken

have three and the viviparous marsupials and eutherians have none.

Spermatozoa. Orthologues of many of the eutherian sperm membrane

proteins related to fertilization40 are present in platypus (and

marsupial) genomes. These include the genes for a number of putative

zona pellucida receptors and proteins implicated in sperm–

oolemma fusion. Testis-specific proteases, which in eutherians participate

in degradation of the zona pellucida during fertilization, are

all absent from the platypus genome assembly.

Monotreme spermatozoa undergo some post-testicular maturational

changes, including the acquisition of progressive motility, loss

of cytoplasmic droplets and aggregation of single spermatozoa into

bundles during passage through the epididymis11. Nevertheless,

maturational changes in the sperm surface that are both unique

and essential in other mammals for fertilization of the ovum have

yet to be identified. Also, the epididymis of monotremes is not highly

adapted for sperm storage as in most marsupial and eutherian mammals.

Consistent with these findings is the absence of platypus genes

for the epididymal-specific proteins that have been implicated in

sperm maturation and storage in other mammals. The most abundant

secreted protein in the platypus epididymis is a lipocalin, the

homologues of which are the most secreted proteins in the reptilian

epididymis41. Notably, ADAM7, a protease that is secreted in the

epididymis of eutherians, has an orthologue in the platypus. This is

a bona fide protease with a characteristic Zn21-coordinating

sequence HExxH in the platypus, in the opossum and the tree shrew

(Tupaia belangeri). However, loss of its proteolytic activity is predicted

in eutherians42 owing to a single point mutation within its

active site (E to Q).

11. Lactation and dentition. Lactation is an ancient reproductive trait

whose origin predates the origin of mammals. It has been proposed

that early lactation evolved as a water source to protect porous

parchment-shelled eggs from desiccation during incubation43 or as

a protection against microbial infection. Parchment-shelled egglaying

monotremes also exhibit a more ancestral glandular mammary

patch or areola without a nipple that may still possess roles in egg

protection. However, in common with all mammals, the milk of

monotremes has evolved beyond primitive egg protection into a true

milk that is a rich secretion containing sugars, lipids and milk proteins

with nutritional, anti-microbial and bioactive functions. In a

reflection of this eutherian similarity platypus casein genes are tightly

clustered together in the genome, as they are in other mammals,

although platypus contains a recently duplicated b-casein gene

(Supplementary Fig. 2).

12. Mammalian casein genes are thought to have originally arisen by

duplication of either enamelin or ameloblastin44, both of which are

tooth enamel matrix protein genes that are located adjacent to the

casein gene cluster in eutherians and, we find, also in platypus. Adult

platypuses, as well as echidnas, lack teeth but the conservation of

these enamel protein genes is consistent with the presence of teeth

and enamel in the juvenile, as well as the fossil platypuses45.

Venom. Only a handful of mammals are venomous, but the male

platypus is unique among them in delivering its poison not via a bite

but from hind-leg spurs. Despite the obvious difficulties in obtaining

samples, it is now known that platypus venom is a cocktail of at least

19 different substances46 including defensin-like peptides (vDLPs),

C-type natriuretic peptide (vCNP) and nerve growth factor (vNGF).

When analysed phylogenetically and mapped to the platypus genome

assembly, these sequences are revealed to have arisen from local

duplications of genes possessing very different functions (Fig. 4).

Notably, duplications in each of the b-defensin, C-type natriuretic

peptide and nerve growth factor gene families have also occurred

independently in reptiles during the evolution of their venom47.

Convergent evolution has thus clearly occurred during the independent

evolution of reptilian and monotreme venom48.

13. Immunity. Although the major organs of the monotreme immune

system are similar to those of other mammals49, the repertoire of

immunity molecules shows some important differences from those

of other mammals. In particular, the platypus genome contains at

least 214 natural killer receptor genes (Supplementary Notes 18)

within the natural killer complex, a far larger number than for human

(15 genes50), rat (45 genes50) or opossum (9 genes51).

Both platypus and opossum genomes contain gene expansions in

the cathelicidin antimicrobial peptide gene family (Supplementary

Fig. 3). Among eutherians, primates and rodents have a single cathelicidin

gene52,53, whereas sheep and cows have numerous genes that

have been duplicated only recently54. The expanded repertoire of

cathelicidin genes in both marsupials and monotremes may arm their

immunologically naive young with a diverse arsenal of innate

immune responses. In eutherians, with their increases in length of

gestation and advances in development in utero of their immune

systems, the diversity of antimicrobial peptide genes may have

become less critical. The platypus genome also contains an expansion

in the macrophage differentiation antigen CD163 gene family

(Supplementary Notes 18).

14. Genome landscape

First, we analyse the phylogenetic position of platypus and confirm

that marsupials and eutherians are more closely related than either is

tomonotremes (Supplementary Notes 19).Wethen describe platypus

chromosomes and observe some properties of platypus interspersed

and tandem repeats. We also discuss a potential relationship between

interspersed repeats and genomic imprinting and investigate how the

extremely highG1Cfraction in platypus affects the strong association

seen in eutherians between CpG islands and gene promoters.

Platypus chromosomes. Platypus chromosomes provide clues to the

relationship between mammal and reptile chromosomes, and to the

origins of mammal sex chromosomes and dosage compensation. Our

analysis provides further insight with the following findings: the 52

platypus chromosomes show no correlation between the position of

orthologous genes on the small platypus chromosomes and chicken

microchromosomes; for the unique 5X chromosomes of platypus we

reveal considerable sequence alignment similarity to chicken Z and no

orthologous gene alignments to human X, implying that the platypusX

chromosome evolved directly from a bird-like ancestral reptilian system

55; and the genes on the five platypus X chromosomes appear to be

partially dosage compensated (Supplementary Fig. 5), perhaps parallel

to the incomplete dosage compensation recently described in birds56.

Repeat elements. About one-half of the platypus genome consists of

interspersed repeats derived from transposable elements. The most

abundant and still active repeats are (severely truncated) copies of the

5-kb long-interspersed-element (LINE2) and its non-autonomous

SINE-companion mammalian-wide interspersed repeat (MIR,

Mon-1 in monotremes) that became extinct in marsupials and in

eutherians 60–100 Myr ago. We estimate that there are 1.9 and 2.75

million copies of LINE2 and MIR/Mon-1, respectively, in the 2.3-Gb

platypus genome. DNAtransposons and LTR retroelements are quite

rare in platypus, but there are thousands of copies of an ancient

gypsy-class LTR element (all LTR elements previously identified in

mammals, birds, or reptiles belong to the retrovirus clade). Overall,

the frequency of interspersed repeats (over 2 repeats per kb) is

higher than in any previously characterized metazoan genome.

Population analysis using LINE2/Mon-1 elements distinguished

the Tasmanian population from three other mainland clusters

(Supplementary Fig. 4a, b), in good agreement with tree-based

analysis, physical proximity and previous knowledge of platypus

population relationships57.

Cluster analysis of all LINE2 copies revealed a phylogenetic relationship

lacking branches, as if a single-locus, fast-evolving gene has

steadily spread an exceptional number of pseudogenes over time

(Supplementary Fig. 6). This ‘master gene’ appearance is, to a lesser

degree, also observed for LINE1 in eutherians58, but not to the same

extent for MIR/Mon-1 or other retrotransposons in mammals. The

phylogeny of LINE2 and Mon-1 was also supported by a genomewide

transposition-in-transposition (TinT) analysis59 (Supplementary

Tables 7 and 8). LINE2 density is similar on all chromosomes

(Supplementary Fig. 7); it does not correlate with chromosome

length (and recombination rate) as the CR1 LINE density does in

the chicken genome19, nor is it higher on sex chromosomes than on

autosomes, as LINE1 density is in eutherians (which has led to postulations

on a function in dosage compensation)60.

We compared microsatellites in the platypus genome with those of

representative vertebrates (Supplementary Notes 22). The mean

microsatellite coverage of platypus genomic sequences assembled

into chromosomes is 2.6760.34%; significantly lower than all other

mammalian genomes sequenced so far and most similar to that

observed in chicken (Supplementary Fig. 8). Microsatellites are on

average shorter in platypus than in other genomes (Supplementary

Table 9), but microsatellite coverage surpasses chicken owing to very

long tri- and tetranucleotide repeats (Supplementary Fig. 9). The

platypus has a higher proportion of microsatellites with high A1T

content, in comparison to the other vertebrates examined, an abundance

distribution that has more in common with reptiles than with

mammals (Supplementary Fig. 10).

15. Genomic imprinting. Genomic imprinting is an epigenetic phenomenon

that results in monoallelic gene expression. In the vertebrates,

imprinting seems to have evolved recently and has only been

confirmed in marsupials and eutherian mammals61,62. The autosomal

localization of some imprinted orthologues in platypus is known63.

However, we examined the conservation of synteny and the distribution

of retrotransposed elements in all orthologous eutherianimprinted

clustered and non-clustered genes in the platypus genome.

A representative cluster is shown in Fig. 5 (see also Supplementary

Fig. 12).

Clusters that became imprinted in therians (with the exception

of the Prader–Willi–Angelman locus64) have not been assembled

recently and reside in ancient syntenic mammalian groups, although

some regions have expanded by mechanisms such as gene duplication

or transposition. There were significantly fewer LTR and DNA

elements across all platypus orthologous regions relative to eutherian

imprinted genes (P,0.04 and 0.04, respectively), whereas there was

a significant increase in the sequences masked by SINEs (P,0.03).

The chicken had fewer total repeats and no SINEs or sRNAs.

Comparison of all regions in the platypus with the orthologous

regions in opossum, mouse, dog and human demonstrates that accumulation

of LTR, DNA elements, and simple and low complexity

repeats coincides with, and may be a driving force in, the acquisition

of imprinting in these regions in therian mammals.

The CpG fraction. The eutherian and chicken genomes generally

average around 41% G1C content, although many intervals differ

substantially from the average, particularly in humans (Supplementary

Notes 23). In contrast, the platypus genome averages

45.5% G1C content and rarely deviates far from the average. The

opossum genome averages only 38% G1C content and also has a

narrow distribution (Supplementary Fig. 13). The source of the elevated

G1C fraction in platypus remains unclear. It is explained only

in part by monotreme interspersed repeat elements, as platypus DNA

outside of known interspersed repeats is 44.7% G1C. Furthermore,

tandem repeats of short DNA motifs (microsatellites) in platypus

show an A1T bias, as with other mammals. Recombination-driven

biased gene conversion may be a factor, in agreement with what has

been shown for eutherians65 and marsupials66. This is suggested by

the observation that the six platypus chromosomes where the currently

mapped DNA sequence averages over 45% G1C content (that

is, 17, 20, 15, 14, 10 and 11 in order of decreasing G1C fraction) are

among the 10 shortest (Supplementary Fig. 14), because short chromosomes

have a higher recombination rate67. However, a direct test

is currently lacking because platypus recombination rates have not

been measured. A further examination of the CpG fraction, that

associated with promoter elements, is found in Supplementary

Notes 24 and Supplementary Fig. 15.

Conclusions

The egg-laying platypus is a remarkable species with many biological

features unique among mammals. Our sequencing of the

platypus genome now enables us to compare its sequence characteristics

and organization with those of birds and therian mammals

in order to address the questions of platypus biology and to

date the emergence of mammalian traits. We report here that

sequence characteristics of the platypus genome show features of

reptiles as well as mammals.

Platypus contains a largely standard repertoire of non-proteincoding,

ncRNAs, except for the snoRNAs, which exhibit a marked

expansion associated with at least one retrotransposed subfamily.

Some of these retrotransposed snoRNAs are expressed and thus

may have functional roles. The platypus has fully elaborated

piRNA and miRNA pathways, the latter including many monotreme-

specific miRNAs and miRNAs that are shared with either

mammals or chickens. Many functional assessments of these novel

miRNAs remain to be carried out and will surely add to our knowledge

of mammalian miRNA evolution.

The 18,527 protein-coding genes predicted from the platypus

assembly fall within the range for therian genomes. Of particular

interest are families of genes involved in biology that links

monotremes to reptiles, such as egg-laying, vision and envenomation,

as well as mammal-specific characters such as lactation,

characters shared with marsupials such as antibacterial proteins,

and platypus-specific characters such as venom delivery and underwater

foraging. For instance, anatomical adaptations for chemoreception

during underwater foraging are reflected in an unusually

large repertoire of vomeronasal type 1 receptor genes. However,

the repertoire of milk protein genes is typically mammalian, and

the arrangement of milk protein genes seems to have been preserved

since the last common ancestor of monotremes and therian

mammals.

Since its initial description, the platypus has stood out as a species

with a blend of reptilian and mammalian features, which is a characteristic

that penetrates to the level of the genome sequence. The

density and distribution of repetitive sequence, for example, reflects

this fact. The high frequency of interspersed repeats in the platypus

genome, although typical for mammalian genomes, is in contrast

with the observed mean microsatellite coverage, which appears more

reptilian. Additionally, the correlation of parent-of-origin-specific

expression patterns in regions of reduced interspersed repeats in

the platypus suggests that the evolution of imprinting in therians is

linked to the accumulation of repetitive elements.

We find that the mixture of reptilian, mammalian and unique

characteristics of the platypus genome provides many clues to the

function and evolution of all mammalian genomes. The wealth of

new findings and confirmation of existing knowledge immediately

evident from the release of these data promise that the availability of

the platypus genome sequence will provide the critically needed

background to inspire rapid advances in other investigations of

mammalian biology and evolution.