OCS Study 

•

MMS 2006-049 

Male-specific AFLP markers reveal 

extreme female-biased sex-ratios in 

the surfgrass Phyllospadix torreyi

(Zosteraceae) 

Final Technical Summary 

Final Study Report 

U.S. Department of the Interior 

Minerals Management Service

Pacific OCS Region 
OCS Study 

MMS 2006-049 

Male-specific AFLP markers reveal 

extreme female-biased sex-ratios in 

the surfgrass Phyllospadix torreyi

(Zosteraceae) 

Final Technical Summary 

Final Study Report 

Authors 

Douglas S. Bush 

Daniel C. Reed 

Sally J. Holbrook 

Scott A. Hodges 

Principal Investigators 

Prepared under MMS Cooperative 

Agreement No. 14-35-0001-31063 

by 

Coastal Marine Institute 

Marine Science Institute 

University of California 

Santa Barbara, CA 93106-6150 

U.S. Department of the Interior 

Minerals Management Service Camarillo

Pacific OCS Region June 2006
Disclaimer 

This report has been reviewed by the Pacific Outer Continental Shelf Region, Minerals Management 

Service, U.S. Department of the Interior and approved for publication. The opinions, findings, 

conclusions, or recommendations in this report are those of the authors, and do not necessarily reflect 

the views and policies of the Minerals Management Service. Mention of trade names or commercial 

products does not constitute an endorsement or recommendation for use. This report has not been 

edited for conformity with Minerals Management Service editorial standards. 

Availability of Report

Extra copies of the report may be obtained from: 

U.S. Dept. of the Interior 

Minerals Management Service 

Pacific OCS Region 

770 Paseo Camarillo 

Camarillo, CA 93010 

Phone: 805-389-7621 

A PDF file of this report is available at: 

http://www.coastalresearchcenter.ucsb.edu/CMI/ 

Suggested Citation 

The suggested citation for this report is: 

Bush, D.S., D.C. Reed, S.J. Holbrook and S.A. Hodges. Male-specific AFLP markers reveal extreme 

female-biased sex-ratios in the surfgrass Phyllospadix torreyi (Zosteraceae). MMS OCS Study 2006-

049. Coastal Research Center, Marine Science Institute, University of California, Santa Barbara, 

California. MMS Cooperative Agreement Number 14-35-0001-31063. 17 pages. 
Table of Contents 

FINAL TECHNICAL SUMMARY....................................................................... 1 

FINAL STUDY REPORT...................................................................................... 3 

 Introduction ...................................................................................................... 3 

Methods ............................................................................................................. 4 

 Study organisms ........................................................................................... 4 

 Collection of samples ................................................................................... 4 

 DNA extraction & AFLP amplification ....................................................... 5 

 Cloning and sequencing of male markers .................................................... 5 

 Determination of genotypes ......................................................................... 6 

Results................................................................................................................ 6 

 Identification of male markers ..................................................................... 6 

 Sequence analysis of male markers .............................................................. 8 

 Determination of population sex-ratios ........................................................ 9 

Discussion .......................................................................................................... 10 

 

ACKNOWLEDGEMENTS.................................................................................... 13 

REFERENCES........................................................................................................ 14

APPENDICES ........................................................................................................ 17 

 

 
List of Tables

Table 1. The frequency of AFLP male markers .................................................... 6 

Table 2. Characterization of ten male-specific AFLP markers ............................. 9 

Table 3. The frequency of males in 22 natural populations .................................. 10 

List of Figures 

Figure 1. Map of Santa Barbara, California County coastline ............................... 5 

Figure 2. The frequency of AFLP male markers .................................................... 7 

Figure 3. The frequency of co-occurrence of the ten male markers ....................... 8 

List of Appendices 

Appendix 1. Frequency of male genotypes ............................................................ 17 
Male-Specific AFLP Markers Reveal Extreme Female-Biased Sex-Ratios 

1 

FINAL TECHNICAL SUMMARY 

STUDY TITLE: Population genetics of surfgrass (Phyllospadix torreyi) for use in 

restoration 

REPORT TITLE: Male-specific AFLP markers reveal extreme female-biased sex-ratios in 

the surfgrass Phyllospadix torreyi (Zosteraceae) 

CONTRACT NUMBER: 14-35-0001-31063 

SPONSORING OCS REGION: Pacific 

APPLICABLE PLANNING AREA(S): Southern California 

FISCAL YEAR(S) OF PROJECT FUNDING: FY 00/01, FY 01/02, FY 02/03 

COMPLETION DATE OF REPORT: June 2007 

COST(S): FY 00/01: $67,067; FY 01/02: $53,451; FY 02/03: $8,461 

CUMULATIVE PROJECT COST: $128,979

PROJECT MANAGER: Russell J. Schmitt 

ADDRESS: Coastal Research Center, Marine Science Institute, University of California, 

Santa Barbara, CA 93106-6150 

PRINCIPAL INVESTIGATORS: Douglas S. Bush1

, Daniel C. Reed2, Sally J. Holbrook1,2 

and Scott A. Hodges1* 

ADDRESSES: 1 Department of Ecology, Evolution and Marine Biology, University of 

California, Santa Barbara, CA 93106-9610; 2 Marine Science Institute, University of 

California, Santa Barbara, CA 93106-6150; *Author for correspondence

KEY WORDS: intertidal ecology, sex determination, male/female ratio, genetic markers 

BACKGROUND: The surfgrass Phyllospadix torreyi is a clonal marine angiosperm that 

occurs in dense persistent stands in the intertidal and shallow subtidal zones of moderate to 

high energy coastlines of the northern Pacific Ocean. Surfgrasses are among the most 

productive seagrass communities yet studied and their stands form the basis for a thriving 

intertidal community. Because surfgrass populations have been and may be disturbed through 

oil and gas exploration, understanding how to restore populations is an important goal. 

Complicating these efforts is the fact that surfgrass is a dioecious species consisting of 

separate male and female plants. Natural populations appear to be strongly biased towards 

females and thus replicating sex-ratios may be important in restoration efforts. 
Final Study Report – Hodges et. al. 

2 

OBJECTIVES: Our goal was to determine if sex determination in surfgrass was due to 

genetic factors and then to determine the actual frequency of male and female individuals. 

Because surfgrass forms dense mats it is impossible to identify genetic individuals. 

Furthermore, we sought to determine if there are microhabitat correlates with where each sex 

occurs. 

DESCRIPTION: Using a DNA fingerprinting technique, amplified fragment length 

polymorphisms (AFLP), we scanned the genome of known males and females to identify 

genetic markers associated with sex. We also used these AFLP markers to identify unique 

genetic individuals in random samples. 

STUDY RESULTS: We identified a number of genetic markers that were highly correlated 

with sex. Using a combination of these markers we were able to conclusively sex individuals 

that were non-flowering. All of the markers we identified were male-specific markers 

suggesting that surfgrass has an XY sex-determination system. The genetic markers also 

reveal that the male-determining Y region is likely accumulating transposable elements as has 

been hypothesized for the evolution of Y-chromosomes. After identifying unique genetic 

individuals from random samples across the Santa Barbara Co. coastline, we determined that 

the frequency of males was only about four percent. We did not find any correlation with the 

frequency of males and habitat. 

SIGNIFICANT CONCLUSIONS: We conclude that individual sexes in surfgrass are 

determined by an XY genetic system. Significantly, we find that males of surfgrass are 

exceptionally rare. This suggests that great care should be used to establish male plants during 

restoration to ensure that the population can sustain reproduction. 

STUDY PRODUCTS: 

Published work: 

Bush, DS, DC Reed, SJ Holbrook and SA Hodges. Male-specific AFLP markers reveal 

extreme female-biased sex-ratios in the surfgrass Phyllospadix torregyi (Zosteraceae). 

submitted, Molecular Ecology. 

Presentations: 

Bush, DS, DC Reed, SF Holbrook and SA Hodges. 2002. Sex-specific markers in surfgrass, 

Phyllospadix torreyi and extreme female-biased sex ratios. Meeting on “Sex-specific genetic 

markers”, University of Lausanne, La Sage, Switzerland. 
Male-Specific AFLP Markers Reveal Extreme Female-Biased Sex-Ratios 

3 

 FINAL STUDY REPORT

Introduction 

Dioecious species are predicted to have unbiased sex-ratios due to negative frequency 

dependent selection (Fisher 1930). In dioecious plants however, equal sex-ratios account for 

only 29 percent of species and 57 percent of species have male-biased ratios (Delph 1999). A 

number of factors have been suggested to account for male-biased sex-ratios, in particular, 

differences in the cost of reproduction between the sexes (Lloyd & Webb 1977). If females 

invest a higher proportion of resources to reproduction than males, trade-offs in other lifehistory traits can result. For instance, stress may cause females to have higher mortality 

(Lloyd 1974, Meagher 1981) or flower less frequently (Cipollini & Stiles 1991). 

In contrast to male-biased sex-ratios, female-biased sex-ratios are rare in plants, accounting 

for only 14 percent of species (Delph 1999). However, among plants with sex chromosomes, 

female-biased sex-ratios are particularly common (Lloyd 1974). A number of hypotheses 

have been developed to account for this genetic association including selfish genetic elements 

(Taylor 1999), the relative competitive ability of pollen tubes carrying different sex 

chromosomes, termed certation (Correns 1917, Stehlik & Barrett 2006), as well as local 

competition (de Jong & Klinkhamer 2002) or differential herbivory between the sexes (Agren 

et al. 1999). For example, in Silene latifolia, sex-ratio distorters have been identified (Taylor 

1994, 1999) and in Rumex, strong evidence for certation has recently been reported (Stehlik & 

Barrett 2006). 

The association between sex chromosomes and female-biased sex-ratios suggests that the sex 

chromosomes themselves cause the lower frequency of males. In most plants studied to date 

with sex chromosomes, males are the heterogametic sex though many dioecious species have 

not been studied (Charlesworth 2002, Vyskot & Hobza 2004). Because the Y chromosome 

does not recombine with the X, the Y chromosome can accumulate deleterious mutations. 

This accumulation is accelerated due to the lower effective population size of the Y 

chromosome compared to autosomes and results in degeneration of gene function (Rice 1987, 

Charlesworth 2002, Vyskot & Hobza 2004). This degeneration of the Y-chromosome could 

then lead directly to impaired performance of males (Charlesworth 2002) and may account for 

the poorer performance of Y-bearing pollen (Stehlik & Barrett 2006). 

Among the few reports of strong female-biased sex-ratios in plants, those for the marine 

angiosperm surfgrass (Phyllospadix torreyi) are exceptional (Dudley 1893, Cox et al. 1992, 

Williams 1995). Williams (1995) reported a population average of approximately 85 percent 

females on Santa Catalina Island, California. However, these numbers were based on the 

number of observed flowering shoots and may be biased by a number of factors. Phyllospadix 

torreyi grows as a rhizome and forms dense clonal mats, which over time can become 

fragmented due to disturbance making the identification of single individuals essentially 

impossible. Thus, reported population sex-ratios based on flowering represent data on ramets 

rather than genetic individuals. Problems associated with using flowers to estimate population 

sex-ratios will be exacerbated if the sexes differ in the extent of clonal growth, mortality, age 

of reproduction, or the frequency and magnitude of flowering (Webb 1999). Without 
Final Study Report – Hodges et. al. 

4 

knowledge of the identity of unique genotypes, it is impossible to determine the sex-ratio of 

adult plants. 

While Phyllospadix torreyi populations appear strongly female-biased, it is equivocal whether 

this species possesses sex chromosomes (Stewart and Rudenberg 1980). The lack of clear 

evidence of sex chromosomes in P. torreyi is interesting because in plant species with sex 

chromosomes, both the X and the Y-chromosomes are usually larger than the autosomes 

(Parker 1990) and therefore should be easy to recognize. The reported extreme female-biased 

sex-ratios and the lack of clear evidence for sex chromosomes provided the underlying 

motivation for our study. We sought to identify genetic markers that are linked to gender 

determination. Once we identified male-specific markers for P. torreyi we used them to 

determine the sex-ratio of genetically distinct individuals in a large number of natural 

surfgrass populations. 

Methods 

Study organism: The surfgrass Phyllospadix torreyi (Family Potamogetonaceae; den Hartog 

1970) is a clonal marine angiosperm that occurs in dense persistent stands in the intertidal and 

shallow subtidal zones of moderate to high energy coastlines of the northern Pacific Ocean 

(Phillips 1979, Turner 1985, Stewart 1989). Surfgrasses are among the most productive 

seagrass communities yet studied (Ramirez-Garcia et al. 1998, 2002) and are unique among 

seagrasses in that they grow on rocks in wave-swept areas (Phillips & Menez 1988). Their 

thin leaves and flowering shoots emerge from creeping rhizomes that are solidly attached to 

the hard substrate by long branching root hairs, following germination and early growth 

during a period of attachment to a host alga (Blanchette et al. 1999). Phyllospadix is 

dioecious; both male and female plants produce long reproductive shoots (rhapidia) that 

support spikes of flowers (spadices), which are most prevalent during the summer and early 

fall (Williams 1995, authors, unpublished data). 

Collection of samples: Samples obtained from flowering individuals were used to screen for 

gender specific markers in Phyllospadix torreyi (N= 140 male and 280 female). These 

samples were collected from 21 localities distributed along a 260 km stretch of coast near 

Santa Barbara, California, USA. Samples obtained from non-flowering individuals were used 

to genetically determine the population sex-ratios at 22 spatially discrete sites located within a 

159 km segment of the 260 km study region, which encompassed the Point Conception 

biogeographic transition zone (Briggs 1974; Figure 1). 
Male-Specific AFLP Markers Reveal Extreme Female-Biased Sex-Ratios 

5 

Figure 1. Map of Santa Barbara, California County coastline. Locations of study sites in each of the three 

regions are indicated. 

For analysis we divided the 22 sites into three geographic regions: sites north of Point 

Conception (Region 1), sites in the western Santa Barbara Channel (Region 2) and sites in the 

eastern Santa Barbara Channel (Region 3). We collected a total of 591 samples in July 2001 

from the low intertidal zone at each of the 22 sites during low tides and from the shallow 

subtidal zone at 10 of the sites by divers using scuba. 

DNA extraction & AFLP amplification: DNA was extracted from the basal portion of fresh 

leaves using the DNeasy kit (Qiagen). Each extraction was carried out twice as single 

extractions proved to contain inhibitors to the AFLP reactions. The AFLP protocol followed a 

modified version of Vos et al. (1995) with a 5X digestion with EcoRI and MseI and a 

fluorescently labeled E-primer in each selective amplification. Reaction products were 

separated on a LiCor 4200 DNA sequencer and variable bands were scored with SAGA MX 

AFLP scoring software (LiCor). 

Cloning and sequencing of male markers: Once specific AFLP markers were identified, we 

ran the reactions again but used 33P-labeled primers. The reactions were separated on 

polyacrylamide gels, dried and exposed to X-ray film. Subsequently, we extracted the malemarker bands from the gel, recovered the DNA and PCR amplified them using the appropriate 

AFLP primers. The products were then cloned using pGEM T-Easy cloning kit (Promega). 

Colonies with the correct insert size were identified with PCR amplification of colonies using 

M13 primers. The amplifcation products were used directly in simultaneous bi-directional 

sequencing following the protocol for the Thermosequenase kit (Amersham) with internal 
Final Study Report – Hodges et. al. 

6 

primers T7 and pGEM+46 (5’-CCGCGGGAATTCGAT-3’) containing fluorescent IRD 

labels 800 and 700 (Li-Cor), respectively. Sequencing was conducted on a Li-Cor 4200 

sequencer. 

Determination of genotypes: Because Phyllospadix has extensive growth by rhizomes, 

samples collected at any one site may be ramets and thus not genetically distinct individuals. 

To determine the repeatability of AFLP genotyping from single individuals, we collected two 

samples from some ramets, isolated the DNA and ran the AFLP from each sample separately. 

After scoring, we determined the percentage of bands that were scored identically from these 

replicate samples. We then applied this percentage as a cutoff for identifying different 

genotypes among samples from each site. 

Results 

Identification of male markers: We screened known male and female samples for AFLP 

markers that were in high frequency in one or the other sex. We used 20 pairs of primers, 

which generated 450 variable bands. Of these, we found 10 bands that were highly abundant 

in male samples (Table 1) but not in females; we refer to these bands as male markers. 

Table 1. The frequency of AFLP male markers in Phyllospadix torreyi in the three study regions along the coast 

of California. The frequency of each marker is listed as a proportion of individuals having that marker and was 

determined from samples of known males and females collected from the three regions shown in Figure 1. 

Sample sizes (n) are given in parentheses. 

 Region 1 Region 2 Region 3 All Sites 

 Male Female Male Female Male Female Male Female 

MaleMarker 

Number 

Frequency 

(n) 

Frequency 

(n) 

Frequency 

(n) 

Frequency 

(n) 

1 0.57 

(7) 

0.00 

(17) 

0.94 

(18) 

0.00 

(101) 

0.86 

(111) 

0.02 

(138) 

0.86 

(139) 

0.01 

(261) 

2 1.00 

(7) 

0.00 

(16) 

0.84 

(19) 

0.00 

(111) 

0.96 

(117) 

0.00 

(146) 

0.95 

(147) 

0.00 

(273) 

3 1.00 

(8) 

0.00 

(17) 

0.90 

(20) 

0.00 

(109) 

0.99 

(120) 

0.01 

(145) 

0.98 

(153) 

0.01 

(271) 

4 0.50 

(8) 

0.00 

(17) 

0.84 

(19) 

0.00 

(109) 

0.69 

(115) 

0.01 

(150) 

0.67 

(145) 

0.01 

(276) 

5 1.00 

(8) 

0.47 

(7) 

0.89 

(19) 

0.00 

(116) 

0.95 

(115) 

0.03 

(143) 

0.94 

(145) 

0.04 

(276) 

6 0.88 

(8) 

0.00 

(17) 

0.79 

(19) 

0.00 

(109) 

0.90 

(114) 

0.01 

(147) 

0.88 

(144) 

0.00 

(2.73) 

7 1.00 

(7) 

0.00 

(15) 

0.92 

(13) 

0.00 

(62) 

0.96 

(115) 

0.06 

(125) 

0.96 

(139) 

0.03 

(202) 

8 1.00 

(7) 

0.00 

(15) 

0.79 

(14) 

0.00 

(48) 

0.9 

(110) 

0.00 

(122) 

0.89 

(134) 

0.00 

(185) 

9 1.00 

(7) 

0.00 

(15) 

0.93 

(14) 

0.09 

(46) 

0.95 

(111) 

0.00 

(117) 

0.96 

(135) 

0.02 

(178) 

10 0.8 

(5) 

0.00 

(15) 

0.85 

(13) 

0.00 

(48) 

0.93 

(106) 

0.02 

(118) 

0.91 

(121) 

0.02 

(181) 
Male-Specific AFLP Markers Reveal Extreme Female-Biased Sex-Ratios 

7 

We did not find any bands that were abundant in females but not in males. While the male 

markers were at high frequency in males, no single marker was present in every male (Table 

1, Fig. 2). Furthermore, seven of the ten markers were found in some females (Table 1, Fig. 

2). We then determined the total number of male markers present in all individuals where all 

markers had been scored. We found that over 80% of females had none of the male markers 

and never more than two (Fig. 3). In contrast, nearly half of the males had all 10 male markers 

and never fewer than six. Thus, we classified non-flowering individuals as males when they 

had six or more male markers and as female if they had two or less. 

Male Marker 

1 2 3 4 5 6 7 8 9 10

Proportion of individuals

0.0

0.2

0.4

0.6

0.8

1.0

males females

Figure 2. The frequency of AFLP male markers in flowering male and female individuals of Phyllospadix 

torreyi in the Santa Barbara region. The presence or absence of each marker was determined for all samples. 

The sample size for each marker ranged from 130 to 140 for males and 180 to 280 for females. Male marker 

characteristics are shown in Table 1. 
Final Study Report – Hodges et. al. 

8 

Number of male markers

0 1 2 3 4 5 6 7 8 9 10

Proportion of individuals

0.0

0.2

0.4

0.6

0.8

1.0

males 

females

Figure 3. The frequency of co-occurrence of the ten male markers in male and female Phyllospadix torreyi. 

Data are the proportion of male and female individuals having zero to all ten of the male markers. The number 

of male markers was determined for individuals of known gender (as identified by flowers) where all ten male 

markers could be scored. Sample sizes were 112 and 157 for males and females, respectively. 

Sequence analysis of male markers: Male markers varied from 124 to 835 bp in length, 

including the AFLP primers, which added a total of 38 bp to the native sequence (Table 2). 

The native sequences were then subjected to tBlastx searches at the NCBI website. The four 

longest sequences (338 – 797 bp) had very strong hits to retroelements (Table 2). The smaller 

fragments did not have significant hits to the databases. 
Male-Specific AFLP Markers Reveal Extreme Female-Biased Sex-Ratios 

9 

Table 2. Characterization of ten male-specific AFLP markers in Phyllospadix torreyi. The oligonucleotides 

used to generate the AFLP bands that are associated with maleness (AFLP Primers) and the size of the marker in 

basepairs (Size) are given. Size includes the AFLP primers which add a total of 38 bp to the native sequence. 

Significant homologues identified by a search of the NCBI database using the protein translation of the marker 

DNA sequence are listed. 

Male-Marker 

Number 

AFLP 

Primers 

Size Homologues 

1 M-CTG, E-ACT 161 None identified 

2 M-CTT , E-ACA 124 None identified; 98% Identical to Band 3 

3 M-CAT, E-ACA 125 None identified 

4 M-CTC, E-AAC 218 None identified 

5 M-CTC, E-AAG 376 Pol Polyprotein Reverse Transcriptase 

6 M-CTC, E-AAG 835 Pol Polyprotein Retrotransposon 

7 M-CAA, E-AAC 176 None identified 

8 M-CCA, E-AAG 655 Pol Polyprotein Retroelement 

9 M-CAG, E-AAG 513 Pol Polyprotein Retroelement 

10 M-CCC, E-AAC 270 None identified 

Determination of population sex-ratios: We analyzed a total of 594 samples that were 

collected from plants that were not flowering at the 22 sites. We found that replicate samples 

from the same individual resulted in < 5% difference in scoring and thus used this as a 

conservative estimate of the number of genotypes in our sample. Using this cutoff we 

identified a total of 482 genotypes. For these unique genotypes, we then determined the 

number of male-specific bands and assigned gender to each sample as outlined above. We 

identified 21 male plants, representing only 4.36 percent of all genotypes. Chi-Square 

analyses revealed that the proportion of males did not differ significantly among the three 

coastal regions (Χ2

 = 1.9882, DF = 2, P = 0.39) or between the intertidal and subtidal zones 

(Χ2 = 0.0905, DF = 1, P = 0.77; Table 3). 
Final Study Report – Hodges et. al. 

10 

Table 3. The frequency of males in 22 natural populations in (A) the three study regions summed across the 

intertidal and subtidal zones, and (B) the intertidal and subtidal zones summed across the three study regions. 

Values are the number of samples, genotypes and males for each category. The percentages of genotypes that 

were male are shown in parentheses.

(A) 

Region Samples Genotypes Males 

1 153 119 3 (2.4%) 

2 219 190 9 (4.2%) 

3 219 163 10 (6.0%) 

TOTAL 591 472 22 (4.7%) 

(B) 

Depth Samples Genotypes Males 

Intertidal 461 351 17 (4.8%) 

Subtidal 130 121 5 (4.1%) 

TOTAL 591 472 22 (4.7%) 

Discussion 

In this study, we determined the gender of adult plants in populations of the marine 

angiosperm, Phyllospadix torreyi, using a combination of molecular markers. We were able to 

identify the gender of plants, regardless of their flowering state, and found an exceptionally 

high frequency of females (0.96 over all samples), the highest for any reported flowering 

plant. Previously Williams (1995) reported a slightly lower frequency of females (0.86) on 

Santa Catalina Island, California based on flowering shoots. The differences between 

Williams’ findings (1995) and ours may reflect actual differences in sex-ratios among 

populations on Santa Catalina Island and those along the Santa Barbara Co. coast. 

Alternatively, these populations may have similar frequencies of females, but differ in the 

frequency that males and females flower or in the numbers of flowering shoots they produce. 

Regardless, P. torreyi maintains extreme female-biased populations over a wide-geographic 

area. 

One mechanism that can influence adult sex ratios is performance differences between the 

two sexes (Delph 1999). Among dioecious plants, females generally suffer greater mortality, 

presumably at the expense of providing greater resources to their offspring (Delph 1999). 

Interestingly, Williams (1995) found that males of Phyllospadix torreyi were inferior to 
Male-Specific AFLP Markers Reveal Extreme Female-Biased Sex-Ratios 

11 

females in their ability to adhere to rocky substrates. She also found that males increased in 

abundance (based on flowering) in subtidal habitats and concluded that they experienced 

greater mortality in intertidal habitats due to strong wave action. Like Williams (1995), we 

also noted a tendency for a greater frequency of flowering males in subtidal, compared to 

intertidal, habitats near Santa Barbara (Reed & Holbrook, unpub. data). However, using 

genetic markers, we found no difference in the sex-ratio between these two habitats. This 

result suggests that males may not have differential mortality across the habitats but, rather, 

that they may be more likely to flower, or that females are less likely to flower, in the subtidal 

region. 

Though we did not find evidence for differential survival of the sexes in different habitats, 

differential mortality may still contribute to the extreme sex-ratio found in adult plants. 

Information on the sex-ratios of earlier life stages is needed to determine if males have higher 

rates of mortality than females. Only then will it be possible to determine if sex-ratios change 

through time and thus whether there are performance differences between the two sexes. For 

instance, in Rumex nivalis, Stehlik & Barrett (2005) determined that sex ratios became more 

female biased from seed to adult suggesting that males had greater mortality than females. 

Such a finding would impact restoration efforts of Phyllospadix torreyi, particularly if early 

life-history stages are utilized (Bull et al. 2004). Unfortunately, we were unable to amplify 

DNA extracted from seeds of Phyllospadix torreyi and thus could not determine if a similar 

pattern is happening in this species. 

Like most other plant species with a genetic sex-determination system (Charlesworth 2002), 

we found that males of Phyllospadix torreyi are the heterogametic sex. Despite this finding, it 

is equivocal whether P. torreyi has differentiated sex chromosomes. Stewart & Rudenberg 

(1980) found occasional meiotic preparations in P. torreyi with chromosomes that appeared or 

behaved unusually. However all chromosome counts were n = 9 and the authors were 

equivocal whether the unusual preparations were evidence for sex chromosomes (Stewart & 

Rudenberg, 1980). Harada (as reported in Kuo 2001) found that the Japanese species of 

Phyllospadix (P. iwatensis and P. japonicus) had 2n = 16, 17, 18, or 20 which he interpreted 

as a system of multiple sex chromosomes. However, Uchiyama (1993) found that both sexes 

in these two species had 2n = 20. Thus, while male P. torreyi are the heterogametic sex, the 

sex-determining region may well be only a portion of one of the chromosome pairs. 

When heteromorphic sex chromosomes exist in plants, the Y-chromosome is often larger than 

the X (Parker 1990) and this size difference is thought to be due to the accumulation of 

transposable elements in the non-recombining region of the Y-chromosome (Charlesworth et 

al. 2005). Consistent with this hypothesis, we only found high sequence similarities to 

transposable elements for the male-markers in Phyllospadix torreyi. The high frequency of 

transposable elements coupled with the lack of differentiated sex chromosomes suggests that 

Phyllospadix is relatively early in the evolution of sex chromosomes. However, we do not 

know whether frequency of transposable elements in the male determining region is higher 

than that found in the corresponding regions of females or in autosomal regions and thus 

whether the Y-region is accumulating elements at a particularly high rate. Identifying early 

stages in the evolution of sex chromosomes is of wide interest (Liu et al. 2004), and future 

research in this area on Phyllospadix torreyi may prove to be particularly enlightening. Not 
Final Study Report – Hodges et. al. 

12 

only would P. torreyi provide an interesting system for comparison, but its particularly small 

genome size (approximately 370 Mbp/1C, Hodges unpub. data) may be useful for identifying 

the genes underlying sex determination. 

The presence of a genetic mechanism of sex determination in Phyllospadix torreyi suggests 

that this system itself may influence sex ratios. A non-recombining region of a Ychromosome can accumulate deleterious mutations resulting in degeneration of the genes in 

this region (Charlesworth 2002). Such degeneration could occur because of, or in concert with 

the accumulation of transposable elements (as we have found here for P. torreyi) and may 

affect all functions associated with genes in the non-recombining region. Haploid pollen 

carrying a Y-chromosome would be especially affected by such degeneration (Smith 1963, 

Lloyd 1974, Charlesworth 2002). This in turn may make pollen carrying a Y-chromosome 

less competitive (also known as certation) as has been recently shown in Rumex nivalis

(Stehlik & Barrett 2006), another strongly female-biased dioecious plant. In P. torreyi, pollen 

is produced as long strands that are easily tangled and form mats, suggesting that pollen 

competition may be common. In fact, seed/ovule ratios appear to be high as each flower 

contains only a single ovule and fruit set is high (Williams 1995, Reed & Holbrook, unpub. 

data). Controlled pollination experiments would be extremely useful for determining whether 

strong pollen competition occurs in P. torreyi. However, the wave-swept environment where 

this species occurs will make such studies quite challenging. 
Male-Specific AFLP Markers Reveal Extreme Female-Biased Sex-Ratios 

13 

ACKNOWLEDGEMENTS 

We thank M. Anghera for his help and encouragement in all phases of this work. The 

research was supported by the National Science Foundation (OCE 99-82105) and the 

Minerals Management Service of the U.S. Department of the Interior (MMS Agreement No. 

14-35-00011-30761). The views and conclusions contained in this document are those of the 

authors and should not be interpreted as necessarily representing the official policies, either 

expressed or implied, of the U.S. Government. 
Final Study Report – Hodges et. al. 

14 

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Male-Specific AFLP Markers Reveal Extreme 

17 

APPENDIX A

Frequency of male genotypes in the intertidal and subtidal zones at the 22 study sites. Distance - distance (km) of each site from the m

(Purisima Point). Data include two specimens whose habitat status (intertidal, subtidal) is unknown. 

Site Distance Tot # 

Smps 

Tot # Genotypes 

Tot # Males 

 Total # 

Inter Smps 

Tot # Inter Geno 

# Inter Male 

 Tot # Sub 

Smps 

Purisima Point R 0 20 14 0 20 14 0 0 Site 4 S 1 20 15 0 20 15 0 0 Stairs T 2 18 10 0 18 10 0 0 Lompoc Landing Q 4 30 25 0 20 15 0 10 Wall Beach U 6 19 14 0 19 14 0 0 Honda Point P 18 16 13 0 16 13 0 0 Boat House O 28 30 28 3 20 18 3 10 Region 1 Total 153 119 3 133 99 3 20 San Augustine M 61 19 14 0 19 14 0 0 Drakes F 66 20 16 0 20 16 0 0 Alegria A 70 70 65 1 45 42 1 25 Caliente (C) C 72 30 25 1 20 15 1 10 San Onofre N 79 30 24 1 20 15 0 10 Arroyo Hondo B 84 20 17 4 20 17 4 0 Arroyo Quemado (D) D 86 30 29 2 20 20 2 10 Region 2 Total 219 190 9 164 139 8 55 Hendry’s (G) G 130 30 25 3 20 15 0 10 Light House I 132 20 13 2 20 13 2 0 Shore Line SH 133 50 46 0 25 23 0 25 Leadbetter (H) H 135 30 27 2 20 17 2 10 Miramar K 143 30 20 1 20 10 0 10 Loon point J 148 20 13 0 20 13 0 0 Casitas E 158 19 10 1 19 10 1 0 Rincon L 159 20 12 1 20 12 1 0 Region 3 Total 219 166 10 164 113 6 55 
Final Study Report – Hodges et. al. 

18 

The Department of the Interior Mission

As the Nation's principal conservation agency, the Department of the Interior has responsibility for most 

of our nationally owned public lands and natural resources. This includes fostering sound use of our 

land and water resources; protecting our fish, wildlife, and biological diversity; preserving the 

environmental and cultural values of our national parks and historical places; and providing for the 

enjoyment of life through outdoor recreation. The Department assesses our energy and mineral 

resources and works to ensure that their development is in the best interests of all our people by 

encouraging stewardship and citizen participation in their care. The Department also has a major 

responsibility for American Indian reservation communities and for people who live in island territories 

under U.S. administration. 

The Minerals Management Service Mission 

As a bureau of the Department of the Interior, the Minerals Management Service's (MMS) primary 

responsibilities are to manage the mineral resources located on the Nation's Outer Continental Shelf 

(OCS), collect revenue from the Federal OCS and onshore Federal and Indian lands, and distribute 

those revenues. 

Moreover, in working to meet its responsibilities, the Offshore Minerals Management Program

administers the OCS competitive leasing program and oversees the safe and environmentally sound 

exploration and production of our Nation's offshore natural gas, oil and other mineral resources. The 

MMS Royalty Management Program meets its responsibilities by ensuring the efficient, timely and 

accurate collection and disbursement of revenue from mineral leasing and production due to Indian 

tribes and allottees, States and the U.S. Treasury. 

The MMS strives to fulfill its responsibilities through the general guiding principles of: (1) being 

responsive to the public's concerns and interests by maintaining a dialogue with all potentially affected 

parties and (2) carrying out its programs with an emphasis on working to enhance the quality of life for 

all Americans by lending MMS assistance and expertise to economic development and environmental 

protection.