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ENDANGERED SPECIES RESEARCHEndang Species Res
Vol. 43: 375–386, 2020https://doi.org/10.3354/esr01078
Published November 19
1. INTRODUCTION
The term ‘cryptic species’ refers to 2 or more spe-cies that are classified, or have been classified, as asingle nominal species because of their (at leastsuperficially) indistinguishable morphology (Bick-ford et al. 2007). Although this concept has beenrecognised for nearly 300 yr (Winker 2005), it is onlyover the last 2 decades that molecular delimitationmethods have revealed that cryptic species are quitecommon and widespread across most animal phyla(Pfenninger & Schwenk 2007, Adams et al. 2014,Pérez-Ponce de León & Poulin 2016). Cryptic species
require special consideration in conservation plan-ning because a species already considered threat-ened may be composed of multiple species that areeven more rare than previously thought (Bowen et al.1991, Schönrogge et al. 2002, Ravaoarimanana et al.2004, Sugawara et al. 2018, Yan et al. 2018). The dis-covery of cryptic species within a threatened speciesis rare, but has been found in the myrmecophiloushoverfly Microdon mutabilis (Schönrogge et al.2002), lentic salamander Hynobius dunni (Sugawaraet al. 2018), Chinese giant salamander Andrias da -vidianus (Yan et al. 2018), olive ridley sea turtle Lep-idochelys olivacea (Bowen et al. 1991), and Ma lagasy
© The authors 2020. Open Access under Creative Commons byAttribution Licence. Use, distribution and reproduction are un -restricted. Authors and original publication must be credited.
Publisher: Inter-Research · www.int-res.com
*Corresponding author: [email protected]
Cryptic species in a Vulnerable seabird:short-tailed albatross consists of two species
Masaki Eda1,*, Takeshi Yamasaki2, Hiroe Izumi1, Naoki Tomita3, Satoshi Konno3, Miwa Konno3, Hayao Murakami3, Fumio Sato3
1Hokkaido University Museum, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo 060-0810, Japan2Division of Natural History, Yamashina Institute for Ornithology, Konoyama 115, Abiko 270-1145, Japan
3Division of Avian Conservation, Yamashina Institute for Ornithology, Konoyama 115, Abiko 270-1145, Japan
ABSTRACT: The occurrence of cryptic species within a threatened taxon is rare, but where theydo occur, understanding species boundaries is essential for planning an effective conservationstrategy. The short-tailed albatross Phoebastria albatrus is a Vulnerable seabird that mainlybreeds on Torishima and the Senkaku Islands in the western North Pacific. Although it has beentacitly regarded as a single management unit with 2 breeding sites, the species is known to com-prise 2 genetically separated populations (Senkaku-type and Torishima-type). However, morpho-logical examination of birds from both populations has not been conducted owing to the difficultyin accessing the Senkaku Islands. In this study, we examined the morphological differences be -tween immigrants from the Senkaku Islands to Torishima (Senkaku-type) and native birds on Tor-ishima (Torishima-type) and found significant differences in morphological characteristics be -tween the 2 bird types. In general, Torishima-type birds were larger than Senkaku-type birds,whereas Senkaku-type birds had relatively longer beaks. Based on the morphological differencesfound in this study as well as genetic and ecological differences revealed in previous studies, webelieve that Senkaku- and Torishima-type birds should be classified as different cryptic species.To the best of our knowledge, this is the first case of cryptic species being identified in a threat-ened avian species.
KEY WORDS: Birds · Conservation · Cryptic species · Short-tailed albatross · Threatened species
OPENPEN ACCESSCCESS
376
lemur Lepilemur septentrionalis (Ravaoarimanana etal. 2004). These discoveries are essential for planningappropriate conservation strategies because eachdifferent species might have different requirementsfor effective conservation (Schönrogge et al. 2002,Davidson-Watts et al. 2006).
The short-tailed albatross Phoebastria albatrusis a Vulnerable (D2) (BirdLife International 2018)seabird found in the north Pacific Ocean. Althoughseveral million birds bred in at least 14 colonies inthe late 19th century, the number of individualswas drastically reduced owing to feather huntingduring the late 19th and early 20th centuries (Tick-ell 2000, Hasegawa 2003, US Fish and WildlifeService 2008), and they were once declared ex -tinct in 1949 (Austin 1949). In 1951, however, aremnant population was re-discovered at the Tsu -bamezaki colony on Torishima Island. During the1954−1955 breeding season, only 23 birds and 7eggs were observed worldwide, all on Torishima(Ono 1955, Fujisawa 1967). Thereafter, the numberof birds began to increase as a result of conserva-tion efforts, and adult birds were also found onMinamikojima Island, Senkaku Islands in 1971(Research Group for Senkaku Islands at RyukyuUniversity 1971). Furthermore, artificial transloca-tion of chicks from Torishima to Mukojima Islandin the Bonin Islands during the 2008 and 2012breeding seasons resulted in the establishment ofa new colony in the Bonin Islands (Deguchi et al.2017). At the end of the 2013−2014 breeding sea-son, the global population was estimated to be4200 individuals, with 3540, 650, and 10 birds onTorishima, the Senkaku Islands, and the BoninIslands, respectively (Deguchi et al. 2017, BirdLifeInternational 2018). Research on the SenakakuIslands has not been conducted since 2002, mainlybecause of the difficulty in accessing the islandsdue to political reasons.
The short-tailed albatross was tacitly regardedas a single management unit with 2 breeding sites,and international conservation projects consideredits population structure to be of low concern (USFish and Wildlife Service 2008, Deguchi et al.2012, BirdLife International 2017), although previ-ous studies suggest that the species could includecryptic species (Eda 2004, Eda & Higuchi 2012,Eda et al. 2012, 2016). Eda (2004) and Eda et al.(2012) found that the 2 populations of short-tailedalbatross existed ~1000 yr ago, and descendants ofeach population seem to have survived, basicallyon Torishima and the Senkaku Islands. Addition-ally, they revealed that the sequence di vergence
between the 2 clades is greater than that betweenother Diomedeidae sister species (Eda & Higuchi2012), and that strong but incomplete pre-matingisolation was observed between birds fledged onTorishima and those fledged on Senkaku Islandswhen they bred sympatrically (Eda et al. 2016).Thus, in addition to these lines of genetic andbehavioural evidence, morphological examinationof individuals of the 2 types is essential for taxo-nomic re-evaluation of the species (Eda & Higuchi2012, Eda et al. 2016). However, the difficulty inaccessing the Senkaku Islands has preventedexamination.
Recently, bird censuses and genetic analyses re -vealed that immigrants from the Senkaku Islandshave been breeding on Torishima (Eda et al. 2011,2016). Since 1979, attempts have been made toattach at least one leg ring to all albatrosses hatchedon Torishima before they leave the island (Sato 1999,Yamashina Institute for Ornithology 2005). In total,4146 short-tailed albatrosses were ringed from April1992 to March 2014 (Yamashina Institute for Ornithol-ogy 2002−2015), and 4140 nestlings were countedover 23 breeding seasons (from 1991−1992 to 2013−2014) on Torishima (Hasegawa 2015). However, un -ringed subadult plumage birds have been observedevery year since 1996 in the Hatsunezaki colony,which is located in the northwest of Torishima andwas artificially established using decoys and audiodevices (Sato 2009). Moreover, the number of un -ringed birds has increased. As it is unlikely that theseunringed subadult plumage birds hatched on Tor-ishima before 1979 or that they lost their ring(s), theirnatal site is suspected to be the Senkaku Islandsrather than Torishima. This conjecture was sup-ported by genetic analysis, because all of the 10 un -ringed birds captured shared haplotypes from agenetic lineage that was observed in all birds fromthe Senkaku Islands captured before 2002 but thisapplied to only 7.3% of the birds that hatched on Tor-ishima (Kuro-o et al. 2010, Eda et al. 2011, 2016).
In this study, we examined morphological differ-ences between birds from the Senkaku Islands andTorishima for taxonomic re-evaluation of the speciesand to fill the gap of morphological surveys. For thispurpose, ringed and unringed birds were capturedand measured on Torishima, and their mitochondrialDNA (mtDNA) control region (CR) 2 was analysed toconfirm the origin population (= type) for each bird.Results revealed morphological differences betweenthe 2 short-tailed albatross populations. The exis-tence of cryptic species for this Vulnerable seabird isalso discussed.
Endang Species Res 43: 375–386, 2020
2. MATERIALS AND METHODS
2.1. Field studies
We captured adult short-tailed albatrosses in theHatsunezaki colony on Torishima (Fig. 1) during the2012−2013 and 2018−2019 breeding seasons. To re -duce the disturbance to breeding behaviour of birdsin the colony, we carefully selected isolated individu-als as capture targets. Short-tailed albatrosses werecaptured with permission from the Ministry of theEnvironment and the Agency for Cultural Affairs,Government of Japan, and in compliance with theirguidelines. Most birds in the colony were ringed andconfirmed to have fledged from Torishima, whilesome were unringed and suspected to have emi-grated from the Senkaku Islands (Eda et al. 2011).Genetic analysis of 9 of the 14 unringed birds was
performed in a previous study (Eda et al. 2016),which confirmed that they shared the mtDNA CR2haplotypes mainly observed in birds from theSenkaku Islands.
We took a maximum of 26 morphological measure-ments for each bird: total length (TL), half wing span(WS), natural wing (NW), tail length (TAIL), tarsuslength (TAR), total head length (TH), beak to gapelength (Gape), skull width (SK-W), exposed culmen(EC), beak height at base (BH), beak width at base(BW), beak tip to nostrils front length (NF), beak heightat nostrils (NFH), beak width at nostrils (NFW), pre-maxillary nail length (PNL), mandibular nail length(MNL), eye diameter (EYE), wing base to tip length(WB-T), primary base width (PBW), scapular basewidth (SBW), tarsus thickness (TT), tarsus width(TW), web width (WW), web length (WL), ankle toweb length (A-W), and body weight (Table S1,
Fig. S1 in the Supplement at www. int-res. com/ articles/ suppl/ n043 p375 _ supp.pdf), but some morphological variableswere only measured in individualscaptured at a later point in the study.
To conduct a molecular phyloge-netic analysis and molecular sexing,blood samples (~0.5 ml) were takenfrom the cutaneous ulnar vein of thebirds. Blood obtained was preservedin 99.5% ethanol and stored, first in acool dark place (in the field) and thenin refrigerated conditions (in the labo-ratory), until analyses.
2.2. DNA analysis
To reveal the phylogenetic positionof each bird, the mtDNA CR2 domainI (341 bp) sequence was determinedfor all birds as described in Eda et al.(2016). We refer to CR‘2’ owing to theduplication of CR in Diomedeidae in -cluding Phoebastria albatrosses (Abbottet al. 2005, Eda et al. 2010). In brief,whole DNA was extracted from bloodsamples using a Puregene DNA isola-tion kit (QIAGEN), CR2 domain I wasamplified via polymerase chain reac-tion (PCR) using primers Lcon2.dio(Eda et al. 2010) and H454.gr (Babaet al. 2005), and PCR products werecycle-sequenced using an ABI BigDyeTerminator ver. 1.1 Cycle SequencingFig. 1. Short-tailed albatross breeding sites
377Eda et al.: Cryptic species in short-tailed albatross
378
kit (Thermo Fisher Scientific) and run on an ABIPRISM 3130 genetic analyser (Thermo Fisher Scien-tific). The obtained sequences were aligned with 24sequences from 53 P. albatrus individuals sampledon Torishi ma (41 ringed chicks, 9 unringed adults)and the Senkaku Islands (1 chick, 2 adults) (Gen-Bank accession numbers AB254197−AB254240 andLC066675− LC066677) using ClustalX in MEGA7.0 (Kumar et al. 2016). Four sequences of Laysan albatross P. im mutabilis (AB276048−AB276050 andAB276055) and 5 sequences of black-footed albatrossP. nigripes (AB276051, AB276057, AB276059, AB276061, and AB276063) were included as outgroupsequences. A neighbour-joining (NJ) tree with 1000bootstrap re plications was constructed for the ob -tained sequences from each bird and 33 sequencesretrieved from GenBank with MEGA 7.0. To identifythe sex of each bird, we used the CHD gene follow-ing established protocols (Fridolfsson & Ellegren1999).
2.3. Morphological analysis
Morphological differences between sexes and pop-ulations were tested using t-tests, a principal compo-nent analysis (PCA), and discriminant function ana -lysis (DFA). Because the molecular analyses revealedthat captured birds were biased to male and thatfemale birds originating from Torishima were rare(see Section 3.2), morphological sexual differenceswere tested only for birds originating from the Sen -kaku Islands, while morphological population differ-ences were tested only for male birds. To reveal thegeneral morphological similarities and dissimilari-ties between populations and sexes, a PCA was con-ducted using the 9 measurements obtained for allcaptured individuals (WS, TAIL, TAR, TH, Gape, EC,NF, NFH, and NFW), using a correlation matrix. Toreveal the effective discriminant criteria betweenmales from the 2 populations, DFA was conductedusing 3 beak measurements (EC, BH, and NFH) aftera homogeneity test of covariance matrices. The rea-son for this selection was higher applicability for oldskin (all measurements), less overlap between thepopulations (BH and NFH), and proportional differ-ence between the 2 populations (EC) (see Section 3.3).DFA was performed using the linear and quadraticdiscriminant function, as covariance matrices were notsignificantly different among species (see Section 3.3).Leave-one-out classification was performed to testthe robustness of the identification criteria. All sta-tistical analyses were conducted using Systat 13
(Systat Software). All measurements were log trans-formed before conducting the analyses.
3. RESULTS
3.1. Field studies
During the research seasons, we captured 24 (10ringed, 14 unringed) adult short-tailed albatrosses.Although blood was collected from all captured birds,measurements were not completed for some birdsbecause of the bird’s condition and some measure-ments were only taken at a later point in the study(Table 1).
3.2. DNA analysis
The target mtDNA CR2 sequence was determinedfor all bird blood samples, and 12 haplotypes werefound. By comparing these haplotypes with thoseavailable in the GenBank database, we identified 4as newly observed; these were de posited in the data-base with accession numbers LC534780−LC534782.The NJ tree clearly showed that there were 2 majorclades in the short-tailed albatross, which were sup-ported by 80 and 95% bootstrap values, respectively(Fig. 2). All 10 se quences from the ringed birds clus-tered with 18 of 20 sequences from Tsubamezakicolony on Torishima. Thus, birds belonging to theclade are referred to herein as Torishima-type. All se -quences obtained from 5 individuals without ringsclustered with 3 sequences from the Senkaku Islandsand previously analysed 9 unringed birds, and with 2sequences from the Tsubamezaki colony. Thus, birdsbelonging to this clade are referred to herein as theSenkaku-type. Molecular sexing revealed that 10 outof 14 Senkaku-type birds and 8 out of 10 Torishima-type birds were male, and the remainder were female.
3.3. Morphological analysis
After identification and sexing, it was apparentthat 26, 13, 26, and 24 measurements were re cordedfor Senkaku-type males, Senkaku-type females, Tor-ishima-type males, and Torishima-type females, re -spectively (Table 1). For Senkaku-type birds, maleswere larger than females on average for 12 out of 13variables, with TAIL being the exception. Significantsexual morphological differences were revealed in 3
Endang Species Res 43: 375–386, 2020
out of 13 measurements (TL, TAR,and body weight) for Senkaku-type birds, while measurementsfor males and females overlappedfor all measured variables. Asthere were only 2 female Tor-ishima-type birds, their morpho-logical sexual difference couldnot be statistically tested. How-ever, on average, female birdswere smaller than male birds forall 24 measurements. In addition,both female birds were smallerthan the smallest male bird for 15of the 24 measurements. On aver-age, Torishima-type males werelarger than Senkaku-type malesfor 24 of 26 mensural points, withEC and NF being the exceptions.Moreover, TL and body weightvalues of the smallest Torishima-type male bird were higher thanthose of the largest Senkaku-typemale bird. There were significantdifferen ces between Senkaku-and Torishima-type male birds in16 of 26 measurements, with Tor-ishima-type birds being largerthan Senkaku-type birds. Therewere no significant differences inEC and NF (Fig. S2).
PCA showed 2 principal compo-nents (PC) with eigenvalues largerthan 1.0 (Table 2). PC1 and PC2accounted for 51.9 and 20.6% ofthe total variance, respectively.Component loadings for PC1 werepositive in all 9 measurements. Asa higher PC1 score is related to alarger body size, PC1 score wasregarded as an indicator of size.Meanwhile, component loadingsfor PC2 were positive for NF,Gape, TH, and EC. All measure-ments with positive componentloadings were related to the beaklength. Contrastingly, componentloadings were negative for TAR,NFH, WS, TAIL, and NFW, noneof which were related to beaklength but were associated withother body elements or the robust-ness of beak. Therefore, higher
Mal
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emal
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enk
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t-te
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t-te
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ax.
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Min
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vs. S
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in.
Ave
.M
ax.
N
Tot
al le
ng
th (
TL
)89
793
0.7
960
1097
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05.4
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*89
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1.7
915
3*
920
925.
093
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Hal
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ing
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an (
WS
)10
5011
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1200
1011
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81.0
1230
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1035
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264
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1105
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102
Nat
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NW
)51
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Tai
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(T
AIL
)14
3.6
156.
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156.
616
5.4
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150
158.
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Tar
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len
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AR
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100
1010
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Bea
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Sk
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th (
SK
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67.4
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***
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Exp
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e (B
W)
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39.2
41.3
1039
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.644
.28
***
36.4
36.8
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tip
to
nos
trils
fro
nt
len
gth
(N
F)
9310
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108.
610
98.5
102.
710
6.5
899
.210
1.3
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.596
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Bea
k h
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ht
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ostr
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NF
H)
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42.3
45.1
1045
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.948
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39.9
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43.3
440
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Bea
k w
idth
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trils
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dia
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er (
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16.6
20.5
23.1
920
.621
.822
.76
20.6
20.7
20.8
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ing
bas
e to
tip
len
gth
(W
B-T
)97
810
33.2
1090
910
5010
90.3
1180
8*
990
1000
.010
102
Pri
mar
y b
ase
wid
th (
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W)
170
179.
823
510
183
204.
423
58
*17
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7.5
185
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cap
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r b
ase
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th (
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W)
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242.
827
010
238
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428
08
230
240.
025
02
Tar
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thic
kn
ess
(TT
)13
14.9
16.1
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15.3
16.9
814
.915
.215
.52
Tar
sus
wid
th (
TW
)7.
98.
69.
110
8.8
9.3
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58.
68.
72
Web
wid
th (
WW
)11
512
7.1
136
1012
213
5.6
155
811
011
5.0
120
2W
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ng
th (
WL
)13
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8.2
144
714
514
6.7
150
3*
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81
An
kle
to
web
len
gth
(A
-W)
220
231.
524
310
243
250.
326
48
***
225
229.
023
32
Bod
y w
eig
ht
3500
4385
.047
5010
4850
5312
.559
508
***
3600
3875
.043
004
*39
5040
00.0
4050
2
Tab
le 1
. Mea
sure
men
ts (
all i
n m
m e
xcep
t b
ody
wei
gh
t, in
g)
of s
hor
t-ta
iled
alb
atro
ss a
nd
res
ult
s of
t-t
ests
bet
wee
n S
enk
aku
- an
d T
oris
him
a-ty
pe
mal
es (
SM
an
d T
M)
and
bet
wee
n S
enk
aku
-typ
e fe
mal
es (S
F) a
nd
SM
. See
Tab
le S
1 an
d F
ig. S
1 fo
r m
ensu
ral p
oin
ts. *
**p
< 0
.001
; **p
< 0
.01;
*p
< 0
.05;
bla
nk
: not
sig
nif
ican
t
379Eda et al.: Cryptic species in short-tailed albatross
AB254219
RM4
AB254197
AB254199
AB254220
AB254234
RM1
RM8
AB254235
RF1
AB254226
AB254222
RM5
AB254227
AB254205
RM7
AB254218
AB254198
RM3
RF2
AB254211
RM6
AB254228
AB254217
AB254230
RM2
AB254232
AB254221
AB254206
UM8
UM10
LC066676 (UF2, UM4)
UM7
AB254200
LC066677 (UM3)
UM9
LC066675 (UF1)
AB254238 (UF3, UF4, UM1, UM2, UM5)
UM6
57
58
65
99
55
98
99
74
93
99
99
99
62
63
68
95
92
97
61
98
53
79
80
84
95
80
70
87
54
66
62
69
5
Senkaku-type
Torishima-type
Black-footed albatross
Laysan albatross
Fig. 2. Neighbour-joining tree for short-tailed albatross. Laysan and black-footed albatrosses were used as the outgroup. Boot-strap values over 50% obtained from 1000 resamplings are shown. R and U denote samples from ringed and unringed birds,respectively; F and M denote samples from female and male birds, respectively. ABxxxxx: sequences from birds at Tsub-amezaki colony on Torishima; *AB254238: sequence from birds in Senkaku Islands; LCxxxxx: sequences from unringed birds
at Hatsunezaki colony on Torishima
380 Endang Species Res 43: 375–386, 2020
PC2 score indicated relatively longer but thinnerbeaks. The PC scores scatterplot revealed that mostmales (with 2 exceptions in the Senkaku-type) hadpositive PC1 scores, whereas females had negativeones, and most Senkaku-type birds (with 2 excep-tions) had positive PC2 scores, whereas Torishima-type birds had negative ones (Fig. 3).
Males from the 2 populations differed significantlyin 3 beak measurements (BH, EC, and NFH) (Pillai’sTrace = 0.774, F = 11.160, df = 4, 13, p < 0.001). Therewas no significant difference between the covariancematrices for males from the 2 populations (χ2 = 10.79,df = 10, p > 0.05), and the following linear discrimi-nant functions for Senkaku- (FLSM) and Torishima-type males (FLTM) were derived:
FLSM = −28 270.777 + 13 793.542 × BH + 21 067.330 ×EC − 7527.422 × NFH
FLTM = −28 420.975 + 14 051.214 × BH + 20 889.911 ×EC − 7474.601 × NFH
In the leave-one-out classification, 89% of birdswere correctly identified, and Senkaku- and Tor-ishima-type birds were correctly identified with 90%(9 out of 10 individuals) and 88% (7 out of 8 individ-uals) accuracy, respectively.
Quadratic discriminant functions for Senkaku-(FQSM) and Torishima-type male (FQTM) were as follows:
FQSM = −18 142.694 + 10 840.443 × BH + 11 835.836 ×EC − 4675.487 × NFH − 7246.289 × BH2 − 5398.385 ×EC2 − 1264.094 × NFH2 + 4609.896 × BH × EC +2441.798 × BH × NFH + 2151.18 × EC × NFH
FQTM = −115 853.159 + 53 105.462 × BH + 93 836.103 ×EC − 37 254.71 × NFH − 10 994.199 × BH2 − 21 011.092× EC2 − 9316.3 × NFH2 − 19 757.91 × BH × EC +16 532.542 × BH × NFH + 18 436.724 × EC × NFH
In the leave-one out classification using quadraticdiscriminant functions, all males were accuratelyclassified into Senkaku- or Torishima-type. Practi-cally, the following function is useful for the discrim-ination between bird types:
FQSM − FQTM = 97 710.47 − 42 265.02 × BH − 82 000.27 ×EC + 32 579.22 × NFH + 3747.91 × BH2 + 15 612.71 ×EC2 + 8052.21 × NFH2 + 24 367.806 × BH × EC −14 090.744 × BH × NFH − 16 285.544 × EC × NFH
By substituting each log10-transformed measure-ment, the bird is determined as Senkaku- or Tor-ishima-type when the sign is positive or negative,respectively.
4. DISCUSSION
4.1. Morphological differences in the short-tailed albatross
We captured 24 adult short-tailed albatrosses atthe Hatsunezaki colony located on Torishima. Molec-ular sexing revealed that captured birds were biasedto male (18 males and 6 females). Not only the rawmeasurement data but also the PCA, especially PC1,revealed that male birds were larger than femalebirds in both Senkaku- and Torishima-types. How-ever, t-tests revealed significant morphological inter-sexual differences in only TL, TAR, and body weightin the Senkaku-type birds, as t-tests could not beconducted for the Torishima-type birds owing to thesmall number of measured female birds. Morpholog-ical sexual dimorphism, in which males were larger
–2.5
–2.0
–1.5
–1.0
–0.5
0.0
1.0
1.5
2.0
2.5
–3.0 –2.0 –1.0 0.0 1.0 2.0
0.5PC1
PC2
Fig. 3. Principal component analysis on 9 measurements of24 short-tailed albatrosses. Red circles: Senkaku-type birds;blue rectangles: Torishima-type birds. Filled and empty
symbols: males and females, respectively
381Eda et al.: Cryptic species in short-tailed albatross
Mensural points Component loadingsPC1 PC2
Half wing span (WS) 0.635 −0.416Tail length (TAIL) 0.494 −0.406Tarsus length (TAR) 0.610 −0.606Total head length (TH) 0.732 0.435Beak to gape length (Gape) 0.749 0.479Exposed culmen (EC) 0.853 0.420Beak tip to nostrils front length (NF) 0.766 0.512Beak height at nostrils front (NFH) 0.804 −0.429Beak width at nostrils front (NFW) 0.773 −0.323
Eigenvalues 4.67 1.85% of total variance explained 51.94 20.56
Table 2. Principal component scores for the 2 principal com-ponent axes based on 9 measurements of 24 short-tailed
albatrosses
Endang Species Res 43: 375–386, 2020
than females, has also been reported in other alba-tross species (Tickell 2000).
Average values for 24 of the 26 measurementswere larger for Torishima-type males than those forSenkaku-type males, and 16 of them were signifi-cantly different according to t-tests. These resultssuggest that Torishima-type males are larger thanSenkaku-type males in general. In measurements ofshort-tailed albatross carpometacarpi at an archaeo-logical site from ~1000 yr ago, birds from the Tor-ishima-type ancestral population were larger thanthose from the Senkaku-type ancestral population(Eda et al. 2012), suggesting that the body size differ-ence between types has persisted for at least 1000 yr.
In contrast, average EC and NF were longer inSenkaku-type males than those in Torishima-typemales, suggesting that morphological differences arenot proportional. PCA, especially PC2 scores, sup-ported this pattern and revealed that it also appliedto female birds, i.e. not only male but also femaleSenkaku-type birds had higher PC2 scores, whichindicated relatively longer and thinner beaks thanthose of both male and female Torishima-type birds.As beak shape is highly related to feeding behaviourand preference, the difference in beak betweenSenkaku- and Torishima-type birds is likely associ-ated with adaptation to different food and/or envi-ronments. Despite the trend of assortative matingbetween 2 types of birds, ringed and unringed pairslikely raised hybrid chicks on Torishima and BoninIslands (Eda et al. 2016, Deguchi et al. 2017), andhybridisation was confirmed by genetic analysis onBonin Islands (Deguchi et al. 2017). In addition, pairsof Senkaku-type birds yielded chicks on Torishima(Eda et al. 2016). Morphological investigations on birdswith parents of both the Torishima- and Senkaku-type, and on Senkaku-type birds fledged from Tor-ishima require further study.
Because all Torishima-type males had longer TLand heavier body weight than those of all Senkaku-type males, these 2 measurements are useful for dis-tinguishing between the 2 male types. However, thesmall sample size should be considered before draw-ing any significant conclusions, i.e. 8 and 10 malesfor Torishima- and Senkaku-type birds, respectively.Additional samples could show overlap in thesemeasurements between both types. In addition, TL isdifficult to measure for skin, especially for older indi-viduals. Furthermore, body weight shows seasonalchange and cannot be measured after a bird hasbeen stuffed. Therefore, we conducted DFA to revealthe identification criteria between the Senkaku- andTorishima-type male birds. Results of leave-one-out
classification revealed that all individuals were cor-rectly identified by quadratic discriminant functionsof 3 beak measurements, i.e. EC, BH, and NFH, sug-gesting that the morphometric approach is useful fordiscrimination between the 2 types of male birds.Because PCA showed a similar pattern of beak shapedifference between female birds from the 2 types, itwill be possible to establish the morphometric dis-crimination criteria for female birds if we conductadditional female bird measurements.
4.2. Cryptic species in the short-tailed albatross
The morphological differences found in this studysupport the idea of the presence of cryptic species. Ingenetic analysis, mtDNA CR2 sequences of modernshort-tailed albatross from Torishima and the Sen -kaku Islands revealed that there were 2 distinct hap-lotypic clades, and that one was specific to Torishimaand the other was distributed across both regions(Eda et al. 2010, 2011, Kuro-o et al. 2010). This geneticstructure, the co-existence of 2 distinct lineages in aregion, i.e. Phylogeographic Pattern II (Avise et al.1987, Avise 2000), can be explained by 2 populationhistory scenarios: (1) 2 isolated populations withrecent admixture on Torishima, or (2) a populationwith a large evolutionarily effective population sizeand significant gene flow, in which 2 separate an -cient lineages were retained by chance. Contrast-ingly, ancient DNA, stable isotope, and morphomet-ric analyses of zooarchaeological bones from theHamanaka 2 site revealed that birds from differentclades formed different populations at that time(~1000 yr ago), and supports the first scenario, i.e.recent co-existence on Torishima (Eda 2004, Eda etal. 2012).
According to phylogenetic analysis, the sequencedivergence in the cytochrome b region of mtDNAbetween the 2 bird types (0.0061−0.0088) is greaterthan that between other Diomedeidae sister species,including those between the Campbell Thalassarcheimpavida and black-browed T. melanophris alba-trosses (0.0026−0.0079) and between wanderingDiomedea exulans and Amsterdam D. amsterdamen-sis albatrosses (0.0053) (Eda & Higuchi 2012). Camp-bell and black-browed albatrosses are clearly dif -ferent in their iris colour, whereas wandering andAmsterdam albatrosses are obviously different intheir final plumage (Tickell 2000). These facts sug-gest that the divergence between Senkaku- and Tor-ishima-type birds was large enough to accumulategenetic differences affecting phenotypic characteris-
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Eda et al.: Cryptic species in short-tailed albatross
tics. Assuming the evolutionary rate of the thirdcodon of cytochrome b region sequence as 1.58%Myr−1 (Nunn et al. 1996), the genetic divergencebetween the 2 lineages occurred 638 000 yr ago (Eda2018).
From an ecological perspective, birds originatingfrom Torishima and the Senkaku Islands were observedsympatrically on Torishima but paired assortativelywith few exceptions, suggesting that Senkaku- andTorishima-type birds achieved a strong but incom-plete pre-mating isolation (Eda et al. 2016). Incompat-ibility of mating displays and differences in the timingof breeding could be proximate cues that result in thetrend of assortative mating in both bird types (Edaet al. 2016). Some differences in courtship displaysbetween the 2 bird types were observed (F. Sato un-publ. data). In addition, the breeding season onthe Senkaku Islands progresses earlier than that onTorishima, as birds on the Senkaku Islands departedfrom the breeding island ~2 wk earlier than those onTorishima in 2002 (Hasegawa 2006). In articles pub-lished over 100 yr ago, a similar trend was also re-ported for the short-tailed albatross on Kuba-jimaand Uotsuri-jima islands in the Senkaku Islands(Kuroiwa 1900, Miyajima 1900), supporting the hypo -thesis that Senkaku-type birds have an earlier breed-ing season than Torishima-type birds. Differencein breeding phenology is recognised as one of thespeciation mechanisms for seabird species withoutapparent physical barriers to dispersal (Friesen 2015).Furthermore, a lifespan difference in food intake be-tween Senkaku- and Torishima-type birds was sug-gested by carbon and nitrogen stable isotope analysesof zooarchaeological bones (Eda et al. 2012).
Under the general lineage species concept, whichdefines a species as a ‘separately evolving metapop-ulation lineage’, evidence of morphological diagnos-ability, reciprocal monophyly, ecological difference,and intrinsic reproductive isolation support the in -ference of a species boundary (De Queiroz 2007).As mentioned above, Senkaku- and Torishima-typebirds had diagnosable morphological characteris -tics, reciprocally monophyletic mtDNA sequences, aclear trend of assortative mating, and heterochronicbreeding phenology. Despite the absence of physicalbarriers to dispersal, the 2 bird types evolved sepa-rately over the course of ~638 000 yr. Therefore, wepropose that Senkaku- and Torishima-type birdsshould be classified as different cryptic species.
The short-tailed albatross Phoebastria albatrus wasnamed by Pallas in 1769, based on specimens cap-tured in the Kamchatka Sea, recently called the Seaof Okhotsk (Pallas 1769). It is difficult to determine
whether Senkaku- or Torishima-type birds shouldinherit the species name P. albatrus, for 3 reasons.Firstly, the type locality, Sea of Okhotsk, has likelybeen used by both types of birds (Eda et al. 2012, N.Tomita et al. unpubl. data). Archaeological albatrossremains (~1000 yr ago) from the Hamanaka site onRebun Island, located where the Sea of Japan andthe Sea of Okhotsk meet, included bones from bothtypes of birds (Eda 2004, Eda et al. 2012), and light-based geolocator tracking revealed that both types ofbirds visit the Sea of Okhotsk (N. Tomita et al.unpubl. data). Secondly, the original description byPallas (1769) is not sufficient for species identifica-tion. Although Pallas (1769) reported 1 value for eachof 12 morphometric measurements, it is unclearwhether they are comparable with the measure-ments in the present study and whether all of themwere taken from the same individual. Finally, thetype specimens were lost (V. Loskot and S. Frahnertpers. comm.). In addition to albatrus, at least 3 namesfor these species have been coined, i.e. chinensis,brachyura, and derogata. Nomenclatural studies arerequired to determine which type of bird takes thename albatrus and to determine an appropriate namefor the other type.
4.3. Implications for short-tailed albatross conservation
After the extinction declaration in 1949 (Austin1949), only 23 birds were observed on Torishima dur-ing the 1954−1955 breeding season (Fujisawa 1967),and the bird numbers and area of occupancy drasti-cally declined within 3 generations (72.3 yr for thespecies) (Hasegawa & DeGange 1982, BirdLife Inter-national 2018). Therefore, the combined taxon, short-tailed albatross P. albatrus sensu lato, was CriticallyEndangered under the current Red List guideline(IUCN Standards and Petitions Committee 2019) inthe 1954–1955 breeding season. With great conser-vation efforts and considering it as a single manage-ment unit with 2 breeding sites, the species popula-tion in creased thereafter and was listed as Vulnerablein the 2018 IUCN Red List under criteria D2 (BirdLifeInternational 2018). However, the present study re -vealed that the species in cluded cryptic species. Tothe best of our knowledge, this is the first case ofcryptic species being identified in a threatened avianspecies.
When we consider that the species includes crypticspecies, Senkaku- and Torishima-type albatrossesshould be assessed and managed separately. Differ-
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ent cryptic species might require different conserva-tion strategies (Schönrogge et al. 2002), as they maybe exposed to different risks. Potential threats to theSenkaku-type birds are probably posed by theirsmall population size and management problems forresearchers owing to low accessibility due to politicalissues. At the end of the 2013−2014 breeding season,the number of mature birds was estimated to be 1734(Deguchi et al. 2017, BirdLife International 2018).Assuming a similar demographic composition on theislands, the number of mature birds was estimated tobe 1462 and 269 birds on Torishima and the SenkakuIslands, respectively. Thus, the estimated number ofbirds on Senkaku can be close to the limit at which apopulation is classified as 'Endangered' (<250; IUCNStandards and Petitions Committee 2019). A recentincreasing trend of immigrants from the SenkakuIslands may be related to the environmental degra-dation of breeding island(s), or full carrying capacitypreventing juveniles from recruiting to the SenkakuIslands. In addition, long-distance (>1500 km) disper-sal of juvenile birds and a linear relationship betweencolony size and proportion of juveniles recruiting totheir hatching place were observed in wanderingalbatross (Inchausti & Weimerskirch 2002). Thus, col -ony size difference between the Senkaku Islands andTorishima could explain the dispersal trend from theSenkaku Islands. Research is urgently required atthe Senka ku Islands to confirm the number of indi-viduals and explain the increasing dispersal trend.
Torishima is an active volcanic island, and its erup-tion is a potential threat for Torishima-type birds.However, models have shown that even small in -creases in chronic mortality rates, such as those re -sulting from bycatch, would have a greater impacton population trends than stochastic and theoreti-cally catastrophic events, including volcanic erup-tions (Finkelstein et al. 2010). Comparative researchon oceanic distribution between the Senkaku- andTorishima-type birds would be essential for estimat-ing the risk of bycatch for each bird type.
The increasing trend in immigrants from the Sen -kaku Islands could affect the genetic integrity of Tor-ishima-type birds through interspecific hybridisation.Viable F1 hybrids have been reported in interspeciespairs in Diomedeidae, including species pairs ofblack-footed × Laysan albatrosses (Rohwer et al.2014), Campbell × black-browed albatrosses (Mooreet al. 2001), and northern royal Diomedea sanfordi ×southern royal D. epomophora albatrosses (Robert-son 1993). As the genetic distance between theSenkaku- and Torishima-type birds is much smallerthan that between black-footed and Laysan alba-
trosses (Eda & Higuchi 2012, Eda et al. 2012), thedivergence between the 2 types is unlikely to achievecomplete post-mating isolation. In the Bonin Islands,2 types of birds confirmed by genetic analysis pairedand yielded chicks (Deguchi et al. 2017), suggestingthat post-mating isolation mechanisms were not sostrong between these types. Therefore, the robust-ness of pre-mating isolation is thought to have amajor impact on whether the admixture of 2 types ofbirds will proceed. So far, 2 types of birds, discrimi-nated by ring status, have shown a clear trend inassortative mating on Torishima, but 6.8% of theobserved pairs were disassortative (Eda et al. 2016).It would be important to note that 2 sequences fromthe birds hatched at Tsubamezaki colony on Tor-ishima (i.e. ringed birds) clustered with Senkaku-type birds. Thus, some of the ringed birds may bedescendants of Senkaku-type birds that emigratedfrom the Senkaku Islands and bred on Torishima.Future studies analysing the nuclear and mtDNA ofringed birds mating with unringed birds and theirchicks are required to accurately evaluate the assor-tative and disassortative mating rates and to predictwhether species boundaries between these 2 speciesbreak down over time.
The third breeding station, the Bonin Islands, couldsuffer more serious issues in this context. The colonywas artificially established by the translocation andhand-rearing of 69 chicks from the Tsubamezakicolony of Torishima to Mukojima during 2008 and2012, as a joint international conservation project be -tween Japan and the USA (US Fish and WildlifeService 2008, Deguchi et al. 2017). Although the pos-sibility of including cryptic species was suggested inearlier work (Eda 2004), the project progressed with-out genetic assessment of translocated birds owing tothe urgent requirements for establishing a third sta-ble breeding location and the lack of taxonomic in -formation about birds on the Senkaku Islands. Thetranslocated birds would be mainly Torishima-typebirds but could include Senkaku-type birds, whichrepresented ~7% of hatched birds in the Tsubame -zaki colony (Kuro-o et al. 2010). As the hand-rearedbirds may not have the ability to recognise the differ-ent cryptic species and/or their original bird types,they would thus not be able to mate assortatively.Under genetically uninformed management efforts,as a further example, the widespread and CriticallyEndangered Chinese giant salamander, which con-sists of at least 5 species-level lineages, was geneti-cally homogenised in captivity, and hybrid offspringwere released back into the wild (Yan et al. 2018). Toexclude the risk of artificial population admixture, a
Endang Species Res 43: 375–386, 2020
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paring census and genetic monitoring of chicksfledged from the Bonin Islands using mtDNA andmicrosatellite DNA are required. The present studyalso emphasises the need for genetic assessments ofseemingly well-known threatened avian species inconservation initiatives.
Acknowledgements. Special thanks to Hiroyoshi Higuchi(Keio University), who provided valuable comments on thestudy. The information on the lost type specimens of theshort-tailed albatross was provided by Vladimir Loskot(Zoological Institute, Russian Academy of Sciences) andSylke Frahnert (Natural History Museum, Berlin). Insightfulcomments from 2 anonymous reviewers clarified thestrengths and weaknesses of this study. This study was partially financially supported by the Ministry of the Envi-ronment Government of Japan, Tokyo Metropolitan Gov-ernment, US Fish and Wildlife Service, the Mitsui & Co. En -vironment Fund, the Suntory Fund for Bird Conservation,Keidanren Nature Conservation Fund, and JSPS KAKENHIGrant Number JP15K14439.
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Editorial responsibility: Mike Bruford,Cardiff, UK
Submitted: May 13, 2020; Accepted: September 17, 2020Proofs received from author(s): November 16, 2020
