DizygoticDizygotic Twinning in the Hawaiian Monk SealTwinning in the Hawaiian Monk SealJessica U. LopezJessica U. Lopez11, Brenda L. Becker, Brenda L. Becker22, Jennifer K. Schultz, Jennifer K. Schultz33, , TheaThea JohanosJohanos--KamKam22, , LizabethLizabeth KashinskyKashinsky11

(1) Joint Institute for Marine and Atmospheric Research, 1125B Ala Moana Blvd, Honolulu, HI, 96814, USA

(2) NOAA Fisheries, 1601 Kapiolani Blvd Suite 1110, Honoulu, Hawaii, 96814, USA

(3) Hawaii Institute of Marine Biology, P.O. Box 1346, Kaneohe, Hawaii, 96744, USA

Correspondence email: Jessica.Lopez@noaa.gov

ABSTRACT

Twinning is extremely rare in pinnipeds, likely due to the high energy demands of

nursing and the difficulty in caring simultaneously for more than one pup. We explored

the incidence of twinning in the Hawaiian monk seal (Monachus schauinslandi) over a

period of 27 years. Seven sets of putative twins (14 pups) of 4875 total births were

recorded between 1982 and 2008, for a minimum twinning rate of 0.001. Microsatellite

genotyping of four of the seven mother and pups revealed that all four dyads tested were

dizygotic twins, ruling out the possibility of alloparenting, with low probability of false

assignment (P = 0.004 - 0.270). The nursing interval (35-44 days) was not significantly

shorter for twins in comparison to singletons born of the same year and island; however

twins had significantly smaller girths at weaning (1-4 standard deviations lower). Twins

were less likely to survive to weaning and age one than their singleton counterparts:

eight of the 14 pups (57%) survived to weaning; five survived to age one (36%); and

only one (7%) has been confirmed to survive past age one. Twinning appears to be a

maladaptive trait in the Hawaiian monk seal, though captive supplemental feeding could

increase the survival rate of twins and other undersized pups.

111 (9)114 (11)118 (11)105 (8)N/AN/AAve Ag (cm)

126 (7)134 (6)124 (11)125 (7)N/AN/AAve DSL (cm)

46 (7)39 (6)37 (4)39 (8)Unknown41 (2)

For Island and Year:

Ave days nursed

4041625010081% singletons

surviving to age one

50010025N/A50% twins surviving to

age one

931008579100Unknown% singletons

surviving to weaning

50100100250100% twins surviving to

weaning

Table 1: Putative twin dyads listed by mother and pup identification number, year, island, outcome, dorsal straight length (DSL) and axillary girth (AG) at death or weaning compared with

the average (and standard deviation) DSL and AG of singleton births for that island and year and twin survival rates in comparison to singleton survival rates by island and year.

Year 1983 1998 2002 2006 2007 2008

Island FFS Oahu FFS Midway Kure Kure

Mom ID Y190 R003 Y305 Y652 KN88 TS40 B6AN

Pup ID FFS8300EN FFS8300EP RYD1 RYD3 YEXE YEXF YE40 YEXC PO22 PO26 KB22 KB24 KWX1 KW28

Pup Sex M M F M M M F F F F F M F F

Days Nursed 39 39 0 0 <4 <4 37 32-33 42 42 >15 >15 0 44

Outcome Weaned WeanedDied

day 1

Died

day 1

Died

day 1-4

Died

day 1-4Weaned

Died

day 33*Weaned Weaned Weaned Weaned

Died

day 1Weaned

DSL (cm) Not measured 102 98 89 88 122 120 113 114 114 107 99 126

AG (cm) Not Measured 57 49 44 44 97 84 82 89 92 88 49 115

Age last seen (yrs) ≥ 1 ≤ 1 7^ 1 1 ≤ 1 ≤ 1 1 ^

*Died due to lethal shark attack ^Last seen during 2009 field season. Further survival will be determined in future field seasons.

MATERIALS AND METHODS

• Annual population assessment activities occurred in the Northwestern Hawaiian Islands (NWHI) with

opportunistic monitoring in the main Hawaiian islands (MHI) from 1982-2008 (Figure 1).

• Seal identities were determined by hind flipper temple tags, unique applied bleach marks, and/or natural

identifying markings.

• Twinning was inferred when an isolated female was observed attending two young pups of similar size,

often with attached umbilical cords and placentas.

• Weaned pups were tagged with hind flipper temple tags. Tissue plugs for genetic analysis were collected

and measurements of dorsal straight length (DSL) and axillary girth (AG) were collected during tagging.

• Dead pups were necropsied and tissues were collected, along with measurements of DSL and AG.

• Genomic DNA was extracted from all tissue plugs. Amplifications were performed at 17 Hawaiian monk

seal microsatellite loci (Schultz et al. 2009) and one grey seal locus (Allen et al. 1995).

• Genotypes of pups and putative mothers were compared to verify maternity and determine monozygotic or

dizygotic twinship.

• Subsequent survival of pups was determined through population assessment monitoring.

RESULTS

Confirming Maternity and Relatedness:

• Genetic analysis of the four twin dyads tested indicated that all are dizygotic twins. Assuming all observed dyads are twins (including

those for which genetic data was unavailable), the minimum prevalence of twinning is 0.1% of the Hawaiian monk seal pups born

over the past 27 years.

Size and Survival:

• Of 14 pups (seven sets of twins), five were found dead shortly after birth, eight survived to weaning, and one died shortly prior to

weaning. Both twins of a dyad either died or survived to weaning with two exceptions. YEXC (FFS 2002) died from a shark bite after

33 days of nursing and KWX1 (Kure 2008) was found dead at one day old.

• Axillary girth at weaning is a good predictor of survivorship to one year of age (Baker 2008). Of the eight pups that survived to

weaning, seven had smaller AG at weaning than the average singleton born of that island and year. The only twin that weaned at

above average girth (KW28, AG = 115 cm) was raised as a singleton because its twin (KWX1) died at or soon after birth.

• Only five pups survived to year one. Twins surviving to weaning were less likely to survive to age one than their singleton

counterparts (Table 1).

ACKNOWLEDGEMENTS:We thank the numerous field crews for their efforts in the collection of data, particularly S. Canja, S. Farry, J. Henderson, J. Klavitter, L. Laniawe, T. Norris, R. Marshall, J. Jones, A. Dietrich, E. Green, B. Harting, J. Palmer,

and M. Vekasy. Dr. R. Braun Dr. A. Aguirre, C. Vanderlip, A. Marie, and M. Hester conducted necropsies on several of the dead twins. We thank B. Antonelis, C. Littnan, J. Baker, A. Harting for comments. For logistical

support, we thank the Papananaumokuakea Marine National Monument, U.S. Fish and Wildlife Service, and the captain, officers, and crew of the NOAA ships Townsend Cromwell and Oscar Elton Sette. This research was

performed under the following NMFS scientific research and enhancement permits: 413, 482, 489, 889, 848-1335, 848-1695, 848-1365. Funding was provided by the Marine Mammal Commission, (Research grant

#GS00M04PDM0027) and the Northwest Hawaiian Islands Coral Reef Ecosystem Reserve. JKS was supported by an EPA START and an NSF IGERT fellowship (Award # 0549514; PI Bruce A. Wilcox.

Figure 1: Observed incidence of twinning in Hawaiian Archipelago

X2

X3

Twins observed

LITTERATURE CITED:Allen, P.J., W. Amos, P.P. Pomerory, and S.D. Twiss. 1995. Microsatelite variation in grey seals (halichoerus grypus) shows evidence of genetic differentiation between two British breeding colonies. Molecular Ecology 4: 653-662.

Baker. J.D. 2008. Variation in the relationship between offspring size and survival provides insight into causes of mortality in Hawaiian monk seals. Endangered Species Research 5: 55-64. Ely, J. J., W. I. Frels, S. Howell, M. K.

Izard, M. E. Keeling, D. R. Lee. 2006. Twinning and heteropaternity in chimpanzees (Pan troglodytes). American Journal of Physical Anthropology 130:96-102. Hoekstra, C., Z. Z. Zhao, C. B. Lambalk, G. Willemsen, N. G.

Martin, D. I. Boomsma and G. W. Montgomery. 2008. Dizygotic twinning. Human Reproduction Update 14:37-47. Schultz, J. K., J. D. Baker, R. J. Toonen, and B. W. Bowen. 2009. Extremely low genetic diversity in the

endangered Hawaiian monk seal (Monachus schauinslandi). Journal of Heredity 100:25-33.

DISCUSSION

Intervention:

• In three cases observers intervened to reunite one or both pups with the mother. The 2006 Midway twins were fed in a captive setting

for 9.5 months past weaning. Without these interventions, survival to weaning and beyond may have been further decreased.

• Dizygotic twinning appears to be hereditary in mammals, such that mothers of twins are more likely to twin again (Hoekstra et al.

2008; Ely et al. 2006). To our knowledge, no female Hawaiian monk seal has produced multiple sets of twins.

Maternal Investment and Environmental Factors:

• Girth at weaning as it relates to survival of pups can be linked to pre-and post-partum maternal investment as well as the productivity

of the environment during gestation and nursing.

• Mothers of twin dyads were experienced and in the prime of their reproductive years. The ages for four of the adult females at the

time of twinning ranged from 11-19 years old. Five of the twin mothers had a minimum of 3-8 single births prior to twinning, and

five twin mothers had 1-4 single births after producing twins. At least three of the females had a single birth the year before and three

pupped the year following the twinning event.

• We do not find a clear overall trend between ocean productivity and twin survivorship due to the overall rare incidence of twinning.

Our results indicate that twinning is maladaptive in the Hawaiian monk seal. However, optimization of captive

feeding programs may lead to increased survival of twins to age one. Due to its rarity, even successful twinning is

unlikely to have a major impact on the overall reproductive rate of this critically endangered species.

(a) Y305 and pups YEXE and

YEXF (b) YEXE and YEXF prior

to necropsy

FFS 2002

(a)(b)

Y653 and pups YE40 and YEXC

FFS 2002TS40 with pups KB22 and KB24

Kure Atoll 2007