ROBERTS BANK TERMINAL 2 TECHNICAL DATA REPORT · RBT2 – WESA Genetics - 2 - December 2014 Table 1...

ROBERTS BANK TERMINAL 2

TECHNICAL DATA REPORT

Coastal Birds

Genetics of Western Sandpipers Using

the Fraser River Estuary during Northward Migration

Prepared for: Port Metro Vancouver 100 The Pointe, 999 Canada Place Vancouver, BC V6C 3T4 Prepared by: Hemmera Envirochem Inc. 18th Floor, 4730 Kingsway Burnaby, BC V5H 0C6 File: 302-042.02 December 2014

Port Metro Vancouver Hemmera RBT2 – WESA Genetics December 2014

Technical Report / Technical Data Report Disclaimer

The Canadian Environmental Assessment Agency determined the scope of the proposed Roberts Bank

Terminal 2 Project (RBT2 or the Project) and the scope of the assessment in the Final Environmental

Impact Statement Guidelines (EISG) issued January 7, 2014. The scope of the Project includes the

project components and physical activities to be considered in the environmental assessment. The scope

of the assessment includes the factors to be considered and the scope of those factors. The

Environmental Impact Statement (EIS) has been prepared in accordance with the scope of the Project

and the scope of the assessment specified in the EISG. For each component of the natural or human

environment considered in the EIS, the geographic scope of the assessment depends on the extent of

potential effects.

At the time supporting technical studies were initiated in 2011, with the objective of ensuring adequate

information would be available to inform the environmental assessment of the Project, neither the scope

of the Project nor the scope of the assessment had been determined.

Therefore, the scope of supporting studies may include physical activities that are not included in the

scope of the Project as determined by the Agency. Similarly, the scope of supporting studies may also

include spatial areas that are not expected to be affected by the Project.

This out-of-scope information is included in the Technical Report (TR)/Technical Data Report (TDR) for

each study, but may not be considered in the assessment of potential effects of the Project unless

relevant for understanding the context of those effects or to assessing potential cumulative effects.

https://www.ceaa-acee.gc.ca/050/documents/p80054/97463E.pdf

https://www.ceaa-acee.gc.ca/050/documents/p80054/97463E.pdf

Port Metro Vancouver Hemmera RBT2 – WESA Genetics - i - December 2014

EXECUTIVE SUMMARY

Port Metro Vancouver (PMV) is assessing the potential to develop the Roberts Bank Terminal 2 Project

(RBT2 or the Project), a new three-berth marine terminal at Roberts Bank in Delta, B.C. The Project is

part of PMV’s Container Capacity Improvement Program (CCIP), a long-term strategy to deliver projects

to meet anticipated growth in demand for container capacity to 2030.

Hemmera has been retained by PMV to undertake environmental studies related to the Project. This

technical data report describes the results of the western sandpiper (Calidris mauri, hereafter referred to

as WESA) genetics study. WESA are small shorebirds that migrate long distances between breeding

grounds in western Alaska and easternmost Siberia, and wintering sites along the Pacific and Atlantic

coasts of the Americas (Wilson 1994). Roberts Bank and Sturgeon Bank, together with Boundary Bay,

are key stop-over points on the Pacific Flyway for millions of WESA on their spring migration.

Migratory species such as WESA have the potential to exhibit genetic structure meaning that individuals

within a species can be divided into groups or lineages based on their genetic make-up (COSEWIC

2011). Recent studies of non-breeding WESA populations in Mexico showed a modest amount of genetic

structure associated with migratory period and habitat type (Enriquez-Paredes et al. 2012). Presently, no

information exists on whether genetically distinct populations of WESA preferentially use Roberts Bank as

opposed to other parts of the Fraser River estuary (FRE) (Haig et al. 1997, Fernandez and Lank 2006).

The objective of this study was to determine if WESA visiting Roberts Bank are genetically differentiated

from those that visit other areas of the estuary (i.e., Sturgeon Bank or Boundary Bay). The study

consisted of three main components: 1) available literature and data review; 2) WESA sampling; and 3)

WESA genetic analysis.

Results show that little genetic structure was found among WESA using the Roberts Bank, Sturgeon

Bank and Boundary Bay sites within the FRE. There was a high level of variability within each site

(AMOVA), suggesting that many individuals from diverse lineages use the estuary. Levels of genetic

variability, expressed both as haplotype and nucleotide diversity, were highest at Roberts Bank,

suggesting that individuals from a large gene pool visit this site on their annual northward migration.

Genetic lineages of WESA do not appear to be segregating according to site within the FRE during

northward migration; therefore, WESA visiting Roberts Bank do not form a distinct genetic group.

Furthermore, haplotypes of individuals that visit the FRE during northward migration fall within the suite of

haplotypes and nucleotide diversity found in individuals overwintering in Mexico.

Port Metro Vancouver Hemmera RBT2 – WESA Genetics - ii - December 2014

TABLE OF CONTENTS

EXECUTIVE SUMMARY ............................................................................................................................... I

LIST OF ACRONYMS AND SYMBOLS ....................................................................................................... 1

1.0 INTRODUCTION .............................................................................................................................. 1

1.1 PROJECT BACKGROUND ........................................................................................................ 1

1.2 STUDY PURPOSE ................................................................................................................... 1

1.3 WESTERN SANDPIPER GENETICS STUDY OVERVIEW ............................................................... 1

2.0 REVIEW OF AVAILABLE LITERATURE AND DATA ................................................................... 3

2.1 MORPHOLOGICAL AND LIFE HISTORY DIFFERENTIATION IN WESTERN SANDPIPERS ................... 3

2.2 WESTERN SANDPIPERS POPULATION GENETICS ..................................................................... 3

2.3 GENETIC DIFFERENTIATION IN OTHER CALIDRIS SPECIES ........................................................ 4

2.4 SUMMARY ............................................................................................................................. 4

3.0 METHODS ....................................................................................................................................... 5

3.1 STUDY AREA ......................................................................................................................... 5

3.2 TEMPORAL SCOPE................................................................................................................. 7

3.3 STUDY METHODS .................................................................................................................. 7

3.3.1 Field Techniques and Observations ...................................................................... 7

3.3.2 DNA Extraction ....................................................................................................... 8

3.3.3 PCR and DNA Sequencing .................................................................................... 8

3.4 STATISTICAL, CALCULATION, AND MODELING APPROACHES ..................................................... 8

3.4.1 Sequence Editing and Alignment ........................................................................... 9

3.4.2 Diversity Indices ..................................................................................................... 9

3.4.3 Haplotype Frequency Histograms .......................................................................... 9

3.4.4 Phylogenetic Analysis ............................................................................................ 9

3.4.5 Analysis of Population Genetic Structure .............................................................. 9

3.5 DATA QUALITY AND INFORMATION MANAGEMENT .................................................................. 10

3.5.1 Sequence Editing and Comparison with Samples from Mexico .......................... 10

3.5.2 Mexico Haplotype Network and Frequency Histogram........................................ 10

4.0 RESULTS ...................................................................................................................................... 11

4.1 STUDY RESULTS ................................................................................................................. 11

4.1.1 Field Capture ........................................................................................................ 11

4.1.2 Data Analysis ....................................................................................................... 11

Port Metro Vancouver Hemmera RBT2 – WESA Genetics - iii - December 2014

4.1.3 Diversity and Haplotype Frequency ..................................................................... 11

4.1.4 Phylogenetic Information by Sampling Location .................................................. 11

4.1.5 Population Genetic Structure ............................................................................... 12

4.2 COMPARISONS WITH DATA FROM MEXICO ............................................................................. 14

5.0 DISCUSSION ................................................................................................................................. 17

5.1 DISCUSSION OF KEY FINDINGS ............................................................................................. 17

5.2 DATA GAPS AND LIMITATIONS .............................................................................................. 17

5.2.1 Missing Mexico Clade .......................................................................................... 17

5.2.2 Absence of Reference Site Samples ................................................................... 18

5.2.3 Absence of Data for Other Genetic Markers ........................................................ 18

6.0 CLOSURE ...................................................................................................................................... 19

7.0 REFERENCES ............................................................................................................................... 20

8.0 STATEMENT OF LIMITATIONS ................................................................................................... 23

List of Tables

Table 1 Western Sandpiper Genetics Study Components and Major Objectives ........................... 2

Table 2 Breakdown of Sample Sizes and Diversity Information for FRE Sites (Roberts Bank,

Sturgeon Bank and Boundary Bay) .................................................................................. 12

Table 3 FRE Population Pairwise FST Estimates ........................................................................... 15

List of Figures

Figure 1 Study Area and Western Sandpiper Capture Sites at Roberts Bank, Sturgeon Bank and

Boundary Bay ...................................................................................................................... 6

Figure 2 Haplotype Networks of the FRE Haplotypes .................................................................... 13

Figure 3 Haplotype Networks of the FRE with Mexico Haplotypes ................................................. 14

Figure 4 Haplotype Frequency Histogram of 607 bp of mtDNA Control Region Sequence Data

from the FRE (A) and Mexico (B) ...................................................................................... 16

List of Appendices

Appendix A Captured Western Sandpiper Morphometrics

Port Metro Vancouver Hemmera RBT2 – WESA Genetics - 1 - December 2014

LIST OF ACRONYMS AND SYMBOLS

ASY After Second Year

AMOVA Analysis of Molecular Variance

bp Base Pairs

DNA Deoxyribonucleic Acid

Fst Fixation Index

FRE Fraser River estuary

h Haplotype Diversity

mtDNA Mitochondrial DNA

π Nucleotide Diversity

PCR Polymerase Chain Reaction

PMV Port Metro Vancouver

RAPD Random Amplified Polymorphic DNA

RBT2 Roberts Bank Terminal 2 Project

SY Second Year

WESA Western Sandpiper (Calidris mauri)


1.0 INTRODUCTION

This section provides an overview of the study, including project background and study components and

major objectives.

1.1 PROJECT BACKGROUND

The Roberts Bank Terminal 2 Project (RBT2 or Project) is a proposed new three-berth marine terminal at

Roberts Bank in Delta, B.C. that could provide 2.4 million TEUs (twenty-foot equivalent unit containers) of

additional container capacity annually. The Project is part of Port Metro Vancouver’s Container Capacity

Improvement Program, a long-term strategy to deliver projects to meet anticipated growth in demand for

container capacity to 2030.

Hemmera was retained by PMV to undertake environmental studies related to the Project. This technical

data report describes the results of the western sandpiper (Calidris mauri, hereafter referred to as WESA)

genetics study.

1.2 STUDY PURPOSE

WESA are small shorebirds that migrate long distances between breeding grounds in western Alaska and

easternmost Siberia, and wintering sites along the Pacific and Atlantic coasts of the Americas (Wilson

1994). Roberts Bank and Sturgeon Bank, together with Boundary Bay, are key stop-over points on the

Pacific Flyway for millions of WESA on their spring migration.

Migratory species such as WESA have the potential to exhibit genetic structure meaning that individuals

within a species can be divided into groups or lineages based on their genetic make-up (COSEWIC

2011). Recent studies of genetic structure in non-breeding WESA populations in Mexico showed a

modest amount of genetic structure associated with migratory period and habitat type (Enriquez-Paredes

et al. 2012). Presently, no information exists on whether genetically distinct populations of WESA

preferentially use Roberts Bank as opposed to other parts of the Fraser River estuary (FRE) (Haig et al.

1997, Fernandez and Lank 2006). The objective of this study was to determine if WESA visiting Roberts

Bank are genetically differentiated from those that visit other areas of the estuary (i.e., Sturgeon Bank or

Boundary Bay).

1.3 WESTERN SANDPIPER GENETICS STUDY OVERVIEW

The WESA Genetics Study consisted of three main components: 1) available literature and data review;

2) WESA sampling; and 3) WESA genetic analysis. Study components, major objectives and a brief study

overview are provided in Table 1.


Table 1 Western Sandpiper Genetics Study Components and Major Objectives

Study Component Major Objective Brief Overview

1) Available literature and data review

To review available information and state of knowledge regarding WESA genetics and to identify key data gaps and areas of uncertainty within the general RBT2 area.

Key findings and components identified from the literature and data review are summarised in this report. The literature and data review was completed before development of the 2012 field program.

2) Western sandpiper sampling

To capture WESA at three sites (Roberts Bank, Sturgeon Bank, and Boundary Bay) within the FRE and to obtain tissue samples for genetic analysis.

Mist-netting was conducted during the spring northward migratory period in April and May of 2012, and blood samples were collected for genetic analysis.

3) Western sandpiper genetic analysis

To identify genetic units of WESA using the FRE as a migratory stopover site.

DNA1 extraction and mitochondrial DNA

sequencing of blood samples was conducted on samples collected during mist-netting.

1 Deoxyribonucleic acid


2.0 REVIEW OF AVAILABLE LITERATURE AND DATA

The following section provides an overview of available literature and data considered by the WESA

genetics study.

2.1 MORPHOLOGICAL AND LIFE HISTORY DIFFERENTIATION IN WESTERN SANDPIPERS

WESA are small shorebirds that migrate long distances between breeding grounds in western Alaska and

easternmost Siberia, and wintering sites along the Pacific and Atlantic coasts of the Americas (Wilson

1994). Tidal mudflats, salt marshes, and sand beaches on the Pacific Coast (from Washington State to

Peru), the Atlantic Coast (from New Jersey to Surinam) and the Caribbean serve as overwintering sites

(Burger 1984, Wilson 1994). The majority of the population spends the non-breeding period in tropical

areas. WESA show very structured morphological variation across their non-breeding range with

individuals at southern wintering sites having longer wings and bills than those wintering further north

(Nebel 2005, O'Hara et al. 2006). In addition, latitudinal segregation by sex is observed, with males

wintering farther north than females (Fernández and Lank 2006). Life history strategies also differ across

the non-breeding range. Juvenile birds that spend the non-breeding season in Mexico return to the

breeding grounds in their first year of life, whereas those wintering in Panama are more likely to delay

reproduction until the following year (Fernández et al. 2004, O'Hara et al. 2005). It is unknown whether

this structured morphological variation is reflected in genetic differentiation (Lank and Nebel 2006).

2.2 WESTERN SANDPIPERS POPULATION GENETICS

At present, little is known regarding the population genetics of WESA. The first study to investigate WESA

population genetics employed the Random Amplified Polymorphic DNA (RAPD) technique to compare

genetic marker frequencies across one breeding site in Alaska and three wintering and/or migratory

stopover sites (Haig et al. 1997). Of the latter three sites, one site was located on the Pacific Coast in

California, and the remaining two were located on the Atlantic Coast in South Carolina. Although sample

sizes were low (5 to 10 birds per site), results indicated differences in RAPD marker frequencies across

sites (Haig et al. 1997). More recently, analysis of WESA mitochondrial DNA (mtDNA) from birds

overwintering in northern Mexico revealed the presence of two distinct groups of haplotypes (i.e., a group

of genes that is inherited together by an organism from a single parent), termed lineages ‘A’ and ‘B’.

Haplotypes of lineage A were more evenly distributed spatially and temporally, across all habitat types

and the overwintering vs. migratory periods, respectively. In contrast, haplotypes of lineage B were found

only during winter in mangrove and cattail marsh habitats, and resulted in low, but statistically significant

genetic structuring across sites and time-periods (Enríquez-Paredes et al. 2012).Whether individuals

from the two different lineages originated from different breeding areas could not be determined

(Enríquez-Paredes et al. 2012). MtDNA sequencing of additional samples from various sites along the

Pacific Coast and one site on the Atlantic Coast of Mexico also indicates haplotype structure (Enríquez

and Fernández 2010).


2.3 GENETIC DIFFERENTIATION IN OTHER CALIDRIS SPECIES

The degree of population structure evident in a particular species varies considerably across other

calidrid sandpipers (Haig et al. 1997). Dunlin (Calidris alpina), for instance, exhibit marked genetic

differentiation across their circumpolar breeding range, and among wintering and migratory stopover sites

(Wenink et al. 1993, 1994; Wennerberg et al. 1999; Wennerberg 2001). MtDNA analyses revealed the

presence of five distinct lineages in dunlin with statistically significant differences in the presence and

frequencies of haplotype groups among geographic locations (Wenink et al. 1993, Wenink et al 1996;

Wennerberg et al. 1999, Wennerberg 2001). The mtDNA lineages correspond to five subspecies

previously defined on the basis of morphology and molt patterns. Further, there is some correlation

between morphological characteristics and haplotype (Wenink et al. 1993, Wenink and Baker 1996,

Wenink et al. 1996); however, the other morphologically defined subspecies remain unaccounted for by

either mtDNA or other analyses (Wenink et al. 1996, Marthinsen et al. 2007).

Statistically significant population differentiation has also been detected in pectoral and semipalmated

sandpipers (Calidris melanotos and Calidris pusilla, respectively; Haig et al. 1997). In rock sandpipers

(Calidris ptilocnemis), two morphologically defined subspecies showed clear phylogenetic differentiation,

but two others could not be distinguished genetically (Pruett and Winker 2005).

Several calidrid sandpiper species show extremely low levels of population structure with weak or

no statistically significant differentiation (Wennerberg et al. 2002; Bühler and Baker 2005; Rönkä et al.

2008, 2012). While Temminck’s stints (Calidris temminckii) and red knots (Calidris canutus) have low

levels of differentiation, white-rumped sandpipers (Calidris fuscicollis) do not show statistically significant

genetic differentiation despite morphological differences between northern and southern breeding

populations (Wennerberg et al. 2002; Bühler and Baker 2005; Rönkä et al. 2008, 2012).

2.4 SUMMARY

Calidrid sandpipers vary widely in their degree of genetic differentiation. WESA show morphological and

life history differences across their non-breeding range, but the degree to which phenotypic differentiation

is reflected in population genetic differentiation is presently unknown. This uncertainty identified the need

to conduct a WESA genetics study to get a better understanding of WESA populations in the RBT2

study area.


3.0 METHODS

Descriptions of the study spatial and temporal scopes, and field sampling and sample analysis methods

are provided below.

3.1 STUDY AREA

The area of interest for the WESA genetic study is the FRE (Figure 1). For the purpose of this analysis,

the estuary was separated into three sites: Roberts Bank, Sturgeon Bank and Boundary Bay. These

locations were chosen because they collectively form one of the key stop-over sites on the Pacific Flyway

for millions of WESA during northward and southward migration (Catherine Berris Associates Inc. 2010).

The objective of this study was to determine if WESA visiting Roberts Bank could be genetically

differentiated from those that visit other areas of the estuary; therefore, sampling was limited to the FRE.

This page is intentionally left blank.


Figure 1 Study Area and Western Sandpiper Capture Sites at Roberts Bank, Sturgeon Bank and Boundary Bay

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3.2 TEMPORAL SCOPE

WESA genetics studies were intended to assess possible genetic structures of northward migrating

WESA in the FRE. Results described in this report represent genetic variation found during the northward

migration in the spring of 2012 and may be an indication of patterns of genetic differentiation during

subsequent northward WESA migrations. Mist-netting to capture WESA occurred on 10 days over the

three-week northward migratory period from April 19 to May 6, 2012. Netting was conducted on rising and

falling tides corresponding to peak high-tide events under appropriate weather conditions (i.e., under dry

conditions with winds below 30 km/h [<Beaufort 5]). Some netting was conducted during the night due to

tide-height constraints.

3.3 STUDY METHODS

Descriptions of the field sampling and sample analysis methods are provided below.

3.3.1 Field Techniques and Observations

Mist net capture of WESA individuals occurred during the spring 2012 field season, which coincided with

the peak migratory period of WESA. Blood samples from WESA individuals were collected in conjunction

with feather samples for the study of WESA migratory connectivity (Hemmera 2014).

3.3.1.1 Field Sampling

Bird handling and animal husbandry skills were required in this study. All field crew personnel were

qualified and permitted for shorebird handling, and specialists were available throughout the sampling

period to consult on bird handling. Locations of mist nets at each of the sites are depicted in Figure 1.

The timing of WESA migration is stratified by age and sex (Butler et al. 1987, Mathot and Elner 2004);

therefore, three sampling periods were established within the WESA peak migration to capture this

variation. During each sampling period, a per-site quota was set for WESA capture so that an

approximately equal number of individuals were captured throughout the migration

(22±4 individuals/site/period). This system was developed to improve chances that a representative

sample of WESA sex and age classes was collected at each of the three sites during the

migratory period.

Mist nets were set up approximately one hour before the peak high tide on a rising tide or one hour before

exposure of intertidal mudflats on a falling tide. Playbacks of social calls were used during night captures

to draw WESA towards the nets. Nets were monitored continuously and taken down after approximately

three hours or when the daily sample size objectives were reached.

Protocol for bird capture and tissue sampling closely followed that of Enriquez-Paredes et al. (2012).

Upon capture, standard morphological measurements of each bird were taken, including bill length for

sex assignment and plumage coloration for age estimate. Blood samples (30 to 60 µL) for DNA extraction


were drawn from the brachial artery on the wing. Blood sampling protocols are thought to minimally affect

the health of individual birds (Hoysak and Weatherhead 1991). Blood samples were deposited in

‘Queens’ storage buffer (see Seutin et al. 1990) and stored on ice for 4-6 hours until they were brought

back to the laboratory. Samples were stored in the laboratory refrigerator at approximately 3 °C.

3.3.2 DNA Extraction

Samples were sent to Genome Quebec for preparation and sequencing. In total 37 samples from

Boundary Bay, 30 Samples from Roberts Bank and 33 samples from Sturgeon Bank were sent to

Genome Quebec. Three samples from Sturgeon Bank and 7 samples from Boundary Bay were sent to

Genome Quebec as trial samples before the remaining 30 samples from each population were sent for

analysis. To optimise sampling over the entire migratory period, samples were selected from each

sampling location so that there were an approximately equal number of individuals from each sampling

location throughout the duration of the migratory period, with the exception of Roberts Bank, where no

individuals were captured during the early migratory period.

DNA extraction was performed at Genome Quebec using Qiagen DNeasy Blood & Tissue Kit (Qiagen

cat: 69504). The protocol entitled Purification of Total DNA from Animal Blood or Cells (Spin-Column

Protocol) with Nucleated Erythrocytes was used. One modification was made by Genome Quebec upon

receipt of the samples: In the protocol step 1b, 220 µL of the sample was gathered for analysis, taking

into consideration that the storage buffer that the blood samples were delivered in would replace the

Phosphate Buffered Solution buffer in the extraction protocol.

3.3.3 PCR and DNA Sequencing

The polymerase chain reaction (PCR) was conducted on a MasterCycler ProS - Vapo protect from

Eppendorf. Forward and reverse primer sequences were derived from Enriquez-Paredes et al. (2012):

CmCRF-(5’-CCCCCATACTACATACCATC-3’) and CmCRR – (5’-GTCCCACAAGCATTCATT-3’).

Thermal cycling conditions were as follows: initial denature step at 96 °C for 1min, followed by 33 cycles

of 96 °C for 10 seconds, 52 °C for 5 seconds and 72 °C for 1 second with a final denature step of 72 °C

for 30 seconds. Each PCR reaction was created to a total volume of approximately 25 µl per reaction and

included 19 µl ddH2O, 5 µl 5x PCR Buffer, 0.5 µl of dNTP, 0.1 µl TAQ KAPA2G Fast HotStart 5U/µl, 0.1 µl

CmCRF and 0.1 µl CmCRR primers, and 0.1 µl of DNA per reaction. DNA was sequenced on a 3730xl

DNA Analyser from Applied Biosystems.

3.4 STATISTICAL, CALCULATION, AND MODELING APPROACHES

Descriptions of approaches taken for statistical, calculation, and modelling methods are provided below.


3.4.1 Sequence Editing and Alignment

Sequences were edited using Four Peaks v.1.7.2 (Griekspoor and Groothuis 2005), and Mesquite

(Maddison and Maddison 2011), and aligned using Clustal W multiple alignment software (Larkin et al.

2007).

3.4.2 Diversity Indices

Molecular diversity indices were calculated using Arlequin (Excoffier et al. 2005). Indices included mean

haplotype diversity (h; measures the uniqueness of a given haplotype within a sample) and mean

nucleotide diversity (π; average number of nucleotide differences per site measured between any

two individuals). Genetic distance was calculated using Kimura 2 Parameter distance, which uses

information about DNA transitions and transversions to calculate a distance metric (Kimura 1980).

All diversity data and subsequent analyses were conducted first on the full set of samples, including all

individuals, then on a subset of the data which included either, only males, only females or only juveniles.

3.4.3 Haplotype Frequency Histograms

A histogram of the haplotype frequency (relative number of individuals per haplotype) was constructed in

PASW Statistics 18.

3.4.4 Phylogenetic Analysis

Phylogenetic haplotype networks, which outline linkages among mitochondrial variants within the sample,

were constructed in Network 4.6.1.1, using the Median Joining and Maximum Parsimony algorithms

(Bandelt et al. 1999, Polzin and Daneshmand 2003).

3.4.5 Analysis of Population Genetic Structure

Analysis of population genetic structure was conducted using Arlequin (Excoffier et al. 2005). Sequences

were analysed for pairwise population differentiation among sites (Roberts Bank, Sturgeon Bank, and

Boundary Bay) and analysis of molecular variance (AMOVA) was used to determine overall partitioning of

genetic variability within sites and among sites throughout the FRE.

A total FRE-wide sample was analysed, which included all sequences collected as part of this study.

Subsequent analyses of population genetic structure were conducted on subsets of the data that included

males only, females only, ASY (after second year) only and SY (second year) only to determine if genetic

structure could be detected in any of these groups independently. Finally, an AMOVA was used to

investigate whether genetic variability could be partitioned among males and females across the FRE.


3.5 DATA QUALITY AND INFORMATION MANAGEMENT

All data was stored within the Hemmera Geomatics Database system.

3.5.1 Sequence Editing and Comparison with Samples from Mexico

FRE DNA sequences were edited in Four Peaks (Griekspoor and Groothuis 2005). Both forward and

reverse sequence data was utilised for each individual. Forward sequence reads tended to degrade

around 465 base pairs (bp). At this point the upstream (5’) end of the reverse compliment of the reverse

sequence read and the 5’ end of the forward sequence were concatenated into the full sequence (length

607 bp) ensuring that sequences of the highest quality were used in the analysis.

Sequences collected by Enriquez-Paredes et al. (2012) were downloaded from the Genbank database

(Accession Numbers: JF720884 - JF720924) and aligned with FRE data using Clustal W multiple

alignment software (Larkin et al. 2007).

3.5.2 Mexico Haplotype Network and Frequency Histogram

Mexican sequences were used as reference samples for the analysis of FRE WESA genetic data. Upon

alignment of FRE sequences with sequences from Enriquez-Paredes et al. (2012), a haplotype network

was constructed using NETWORK 4.6.1.1, using the Median Joining and Maximum Parsimony algorithms

(Bandelt et al. 1999, Polzin and Daneshmand 2003). In order to compare haplotype frequencies and the

relative diversity in the two datasets, a haplotype frequency histogram was constructed in PASW

Statistics 18, using all available WESA sequence data from Enriquez-Paredes et al. (2012) and from this

study of the FRE.


4.0 RESULTS

This section presents the main findings of the study and provides a comparison of data collected from a

similar study conducted in Mexico.

4.1 STUDY RESULTS

This section discusses the results of the WESA genetics study conducted at the Fraser River estuary.

4.1.1 Field Capture

Overall, 199 WESA individuals were caught, measured, and sampled for blood analysis at Roberts Bank,

Sturgeon Bank and Boundary Bay (Figure 1). Of these, 36 individuals were captured at Roberts Bank,

67 at Sturgeon Bank, and 95 at Boundary Bay. Sampling was approximately evenly spread between the

April 19th and May 6

th 2012 except in the case of Roberts Bank where no individuals were captured on

April 20th or April 22

nd. The first individuals captured from Roberts Bank were caught on April 24 2012.

Males and females were found at all three sites, as were ASY and SY individuals (Appendix A and

Table 2).

4.1.2 Data Analysis

Eighty-eight WESA individuals were sequenced for a 607 bp long section of the mitochondrial control

region (Enriquez-Paredes et al. 2012). Eighteen birds of 30 individuals were reliably sequenced from

Roberts Bank, 37 of 37 individuals were reliably sequenced from Sturgeon Bank, and 33 of 33 individuals

were reliably sequenced from Boundary Bay. .

4.1.3 Diversity and Haplotype Frequency

In total, 34 unique haplotypes (i.e., maternal lineages) were sequenced in the three FRE sites. Five

haplotypes were found either at two or at three sites and the remaining haplotypes were found only at

Roberts Bank (6), Sturgeon Bank (10), or Boundary Bay (13). Haplotype diversity by site (proportion of

the total haplotypes) ranged from 0.59 to 0.79, and was highest at Roberts Bank. Nucleotide diversity

(number of segregating nucleotide sites) ranged from 0.0016 to 0.0018 and was also highest at Roberts

Bank (Table 2).

4.1.4 Phylogenetic Information by Sampling Location

Of the 88 haplotypes sampled in the FRE, the WESA haplotype network showed one high frequency

haplotype that consisted of sequences from over 50% of the individuals sampled (n = 52). The central

common haplotype was found in relatively equal frequencies in all three sites. This common haplotype is

homologous (in part: 607 bp) to the central haplotype in lineage A from Enriquez-Paredes et al. (2012)

(Figures 2 and 3).


4.1.5 Population Genetic Structure

No evidence of population genetic structure was detected when all sequences were compared among

Roberts Bank, Sturgeon Bank, and Boundary Bay sites (Table 2). AMOVA results indicated a large but

statistically non-significant proportion of the total genetic variation could be explained within sites (100%,

P = 0.71). The among-site variability explained a lower, also not statistically significant, proportion of the

total genetic variability (42%, P = 0.71).

When the dataset was broken down into sex and age classes, there was no significant genetic

differentiation among sites in either gender group or age class group. Additionally, approximately 100% of

the total overall variation could be explained by within-population variation in AMOVA analyses of these

groupings. Finally, there was no statistically significant genetic variability between males and females

sampled throughout the FRE.

Table 2 Breakdown of Sample Sizes and Diversity Information for FRE Sites (Roberts Bank, Sturgeon Bank and Boundary Bay)

Site N Gender Age

1 Haplotype Nucleotide

Male Female Unknown SY

ASY

Diversity2

Diversity3

Roberts Bank 18 12 6 0 10 8 0.70 0.002

Boundary Bay 36 18 18 0 14 22 0.66 0.002

Sturgeon Bank 33 14 17 2 4 29 0.59 0.002

Notes: 1

Age: SY = second year; ASY = after second year. 2

Haplotype diversity is the relative proportion of unique haplotypes in the sample. 3

Nucleotide diversity is the relative proportion of nucleotide substitutions in each sequence at each site. Nucleotide diversity values are rounded to the nearest 1/1000

th.


Figure 2 Haplotype Networks of the FRE Haplotypes

Note: The size of each circle represents the frequency of each haplotype, such that larger circles are equal to

higher frequency haplotypes. Haplotype color represents sampling location, where red haplotypes indicate Boundary Bay, yellow haplotypes indicate Roberts Bank, black haplotypes indicate Sturgeon Bank. The lines connecting haplotypes indicate single substitution differences. Open circles represent haplotypes that are missing in the dataset.


Figure 3 Haplotype Networks of the FRE with Mexico Haplotypes

Note: Each circle represents a unique haplotype. The size of each circle represents the frequency of each

haplotype, such that larger circles are equal to higher frequency haplotypes. Haplotype color represents sampling location, where red haplotypes indicate Boundary Bay, yellow haplotypes indicate Roberts Bank, black haplotypes indicate Sturgeon Bank, and blue haplotypes indicated Mexico. The lines connecting haplotypes indicate single substitution differences. Open circles represent haplotypes that are missing in the dataset.

4.2 COMPARISONS WITH DATA FROM MEXICO

Forty-one unique haplotypes (i.e., those found in the 2012 study) from Mexico (Enriquez-Paredes et al.

2012) were downloaded from Genbank. Downloaded sequences contained 685 bp from the mitochondrial

control region.

More individuals were sampled in Mexico (163) than on the FRE (88), and the number of unique

haplotypes varied accordingly (Mexico 41, FRE 34). Nucleotide diversity was on the same order of

magnitude within the two studies, with a mean value of 0.0017 in the FRE and a mean value of 0.005 in

the Mexico sample.


After trimming all sequences from Enriquez-Paredes et al. (2012) to 607 bp, a haplotype frequency

histogram depicting both the FRE haplotypes and the haplotypes from Mexico showed that the most

common haplotype was identical between the two datasets as were the second and third most common

haplotypes; however, only two additional haplotypes were identical between the two datasets. Aside from

the shared haplotypes, each dataset also had many unique or rare haplotypes (Figure 4). In a haplotype

network consisting of Mexico samples (trimmed to 607 bp) and FRE samples, a second lineage that was

present at the Mexican site was missing in the FRE (Figure 3). This lineage is associated with variation in

ecological conditions on the wintering grounds in Mexico (see Enriquez Paredes et al. 2012). Levels of

population structure measured as FST were smaller within the FRE than among the Mexican sites

(Enriquez-Paredes et al. 2012) (Table 3). None of the possible pairwise population genetic differentiation

estimates on the FRE were statistically significantly different from zero.

Table 3 FRE Population Pairwise FST Estimates

Site Sturgeon Bank Boundary Bay

Sturgeon Bank

Boundary Bay 0.004

Roberts Bank -0.005 -0.004

Note: Pairwise FST estimates were not significantly different from zero.


Figure 4 Haplotype Frequency Histogram of 607 bp of mtDNA Control Region Sequence Data from the FRE (A) and Mexico (B)


5.0 DISCUSSION

A discussion of the major results arising from the WESA genetics study and data gaps are provided

below.

5.1 DISCUSSION OF KEY FINDINGS

Little genetic structure was found among Roberts Bank, Sturgeon Bank and Boundary Bay sites, within

the FRE. There was a high level of variability within each site (AMOVA), suggesting that many individuals

from diverse lineages use the estuary. Levels of genetic variability, expressed both as haplotype diversity

and nucleotide diversity were highest at Roberts Bank, suggesting that individuals from a large gene pool

visit this site on their annual northward migration.

Results of this study are consistent with other studies on within-region analysis of shorebird genetic

differentiation (Haig et al. 1997, Álvarez-Sánchez 2011, Enriquez-Paredes et al. 2012). Furthermore,

these results are not unusual, as studies of other calidrid shorebirds also detected low levels of

genetic differentiation among populations (Wennerberg et al. 2002; Bühler and Baker 2005; Rönkä et al.

2008, 2012).

The key findings from this study include:

High genetic diversity within sites, but low genetic differentiation among sites within the FRE,

suggesting that no site is genetically distinct within the estuary.

Highest levels of genetic variability at Roberts Bank, suggesting that a large gene pool of

individuals visit this site during their annual northward migration.

Genetic samples from the FRE had the same level of variability as samples from wintering

grounds in Mexico (Enriquez-Paredes et al. 2012).

One genetically distinct clade in Mexico (lineage B) was absent from the samples collected within

the FRE, suggesting that potentially ecologically differentiated populations in Mexico (i.e., the

bulrush population) (Enriquez-Paredes et al. 2012) did not use the FRE during their northward

migration in 2012.

5.2 DATA GAPS AND LIMITATIONS

This section provides an overview of the data gaps and limitations encountered during the WESA

genetics study.

5.2.1 Missing Mexico Clade

In all haplotypes from the FRE, the substitutions that constitute lineage B in Mexico were not found. This

implies that lineage B likely does not visit the FRE during its migration northward to the breeding grounds.

Other possible explanations include temporal shifts in migration for the missing Mexican clade such that it


passed through the FRE before or after this study captured birds; or it is possible that the missing Mexico

clade exists in very low frequencies relative to the entire population and thus our sampling effort missed

sampling individuals in lineage B purely by chance. The approximately 75 bp that were cleaved from

the 3’ end of the sequences from Mexico did not contain the substitutions that formed lineage B, which

suggests that this lineage may be truly missing from the FRE and not an artifact of sequence alignment

or sampling.

5.2.2 Absence of Reference Site Samples

At present, the only available published data on WESA mtDNA comes from the Enriquez-Paredes et al.

(2012) study in Mexico. Data from other parts of the range are lacking; therefore, it is possible that

stopover sites outside of the FRE region are genetically different.

5.2.3 Absence of Data for Other Genetic Markers

The current study lacks data from multiple nuclear and/or mitochondrial loci. The use of additional loci

would give a broader representation of the patterns of population genetic variability in WESA; however,

Enriquez-Parades et al. (2012) find that the mitochondrial Cytochrome Oxidase 1 locus (used in this

study) was sufficient to detect genetic differentiation in wintering Mexican populations.

Despite the above mentioned limitations, the major objectives as outlined in section one have effectively

been achieved.


6.0 CLOSURE

Major authors and reviewers of this technical data report are listed below, along with their signatures.

Report prepared by: Report peer reviewed by: Hemmera Envirochem Inc. Hemmera Envirochem Inc.

Carson Keever, PhD James Rourke, M.Sc. R.P.Bio. Technical Specialist Coastal Birds Discipline Lead Report peer reviewed by: Simon Fraser University

Ron Ydenberg, PhD Professor, Centre for Wildlife Ecology Director


7.0 REFERENCES

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phylogenies. Molecular Biology and Evolution 16:37-48.

Bühler, D. M., and A. J. Baker. 2005. Population divergence times and historical demography in red knots

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Burger, J. 1984. Shorebirds as marine animals. Pages 83-123 in Shorebirds - breeding behavior and

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Butler, R. W., G. W. Kaiser, and G. E. J. Smith. 1987. Migration chronology, length of stay, sex ratio, and

weight of western sandpipers, (Calidris mauri) on the south coast of British Columbia. Journal of

Field Ornithology 58:103-111.

Catherine Berris Associates Inc. 2010. Roberts Bank and Sturgeon Bank Reach Overview: Phase 2.

Burrard Inlet Environmental Action Program.

Committee on the Status of Endangered Wildlife in Canada (COSEWIC). 2011. Designatable units for

caribou (Rangifer tarandus) in Canada. Ottawa. 88 pp.

Enríquez-Paredes, L. M., C. Vilanova, and G. Fernández. 2012. Genetic diversity and population

structure of wintering western sandpipers from the Sinaloa coast, Mexico. Journal of Field

Ornithology 83:85-93.

Enríquez, L., and G. Fernández. 2010. Genetic identity and population structure of nonbreeding western

sandpiper in México. Page 26 in COS/AOU/SCO 2010 Joint Meeting San Diego, California, USA.

Excoffier L., G. Laval, and S. Schneider. 2005. Arlequin (Version 3.0): an integrated software package for

population genetics data analysis. Evolutionary Bioinformatics Online 1: 47–50.

Fernández, G., and D. B. Lank. 2006. Sex, age and body size distributions of western sandpipers during

the nonbreeding season with respect to local habitat. The Condor 108: 547-557.

Fernández, G., P. D. O'Hara, and D. B. Lank. 2004. Tropical and subtropical western sandpipers (Calidris

mauri) differ in life history strategies. Ornitologia Neotropical 15:385-394.

Griekspoor, A. and Groothuis, T. 2005. 4 Peaks. Version 1.7.


Haig, S. M., C. L. GrattoTrevor, T. D. Mullins, and M. A. Colwell. 1997. Population identification of western

hemisphere shorebirds throughout the annual cycle. Molecular Ecology 6:413-427.

Hemmera. 2014. Roberts Bank Terminal 2 technical data report: Migratory connectivity of western

sandpipers using the Fraser River estuary. Prepared for Port Metro Vancouver. Vancouver, B.C.

Available at: http://www.robertsbankterminal2.com/.

Hoysak, D., and P. Weatherhead. 1991. Sampling blood from birds: a technique and an assessment of its

effects. The Condor 93(3): 746-752.

Kimura, M. 1980. A simple method for estimating evolutionary rates of the base substitutions through

comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111-120

Lank, D. B., and S. Nebel. 2006. Cross-cutting research on a flyway scale – beyond monitoring. Pages

107-112 in Waterbirds around the world (G. C. Boere, C. A. Galbraith, and D. A. Stroud, Eds.).

The Stationery Office, Edinburgh, UK.

Larkin, M. A., G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan, H. McWilliam, F. Valentin, I. M.

Wallace, A. Wilm, R. Lopez, J. D. Thompson, T. J. Gibson, and D. G. Higgins. 2007. ClustalW

and ClustalX version 2. Bioinformatics 23(21): 2947-2948.

Maddison, W. P., and D.R. Maddison. 2011. Mesquite: a modular system for evolutionary analysis.

Version 2.75. Available at http://mesquiteproject.org.

Marthinsen, G., L. Wennerberg, and J. T. Lifjeld. 2007. Phylogeography and subspecies taxonomy of

dunlins (Calidris alpina) in western Palearctic analysed by DNA microsatellites and amplified

fragment length polymorphism markers. Biological Journal of the Linnean Society 92:713-726.

Mathot, K. J., and R. W. Elner. 2004. Evidence for sexual partitioning of foraging mode in western

sandpipers (Calidris mauri) during migration. Canadian Journal of Zoology 82(7):1035-1042.

Nebel, S. 2005. Latitudinal clines in bill length and sex ratio in a migratory shorebird: a case of resource

partitioning? Acta Oecologica-International Journal of Ecology 28:33-38.

O'Hara, P. D., G. Fernández, F. Becerril, H. de la Cueva, and D. B. Lank. 2005. Life history varies with

migratory distance in western sandpipers (Calidris maur)i. Journal of Avian Biology 36:191-202.

O'Hara, P. D., G. Fernández, B. Haase, H. de la Cueva, and D. B. Lank. 2006. Differential migration in

western sandpipers with respect to body size and wing length. The Condor 108:225-232.

Pruett, C. L., and K. Winker. 2005. Biological impacts of climatic change on a Beringian endemic: Cryptic

refugia in the establishment and differentiation of the rock sandpiper (Calidris ptilocnemis).

Climatic Change 68:219-240.


Polzin, T., and S. V. Daneshmand. 2003. On Steiner trees and minimum spanning trees in hypergraphs.

Operations Research Letters 31: 12-20.

Rönkä, A., L. Kvist, J. Karvonen, K. Koivula, V. M. Pakanen, D. Schamel, and D. M. Tracy. 2008.

Population genetic structure in the Temminck's stint Calidris temminckii, with an emphasis on

Fennoscandian populations. Conservation Genetics 9:29-37.

Rönkä, N., L. Kvist, V. M. Pakanen, A. Ronka, V. Degtyaryev, P. Tomkovich, D. Tracy, and K. Koivula.

2012. Phylogeography of the Temminck's stint (Calidris temminckii): historical vicariance but little

present genetic structure in a regionally endangered Palearctic wader. Diversity and Distributions

18:704-716.

Seutin, G., B. White, and P. Boag. 1991. Preservation of avian blood and tissue samples for DNA

analyses. Canadian Journal of Zoology 69:82-90.

Wenink, P. W., and A. J. Baker. 1996. Mitochondrial DNA lineages in composite flocks of migratory and

wintering dunlins (Calidris alpina). The Auk 113:744-756.

Wenink, P. W., A. J. Baker, H. U. Rosner, and M. G. J. Tilanus. 1996. Global mitochondrial DNA

phylogeography of holarctic breeding dunlins (Calidris alpina). Evolution 50:318-330.

Wenink, P. W., A. J. Baker, and M. G. J. Tilanus. 1993. Hypervariable-control-region sequences reveal

global population structuring in a long-distance migrant shorebird, the dunlin (Calidris alpina).

Proceedings of the National Academy of Sciences of the United States of America 90:94-98.

Wenink, P. W., A. J. Baker, and M. G. J. Tilanus. 1994. Mitochondrial control-region sequences in two

shorebird species, the turnstone and the dunlin, and their utility in population genetic studies.

Molecular Biology and Evolution 11:22-31.

Wennerberg, L. 2001. Breeding origin and migration pattern of dunlin (Calidris alpina) revealed by

mitochondrial DNA analysis. Molecular Ecology 10:1111-1120.

Wennerberg, L., N. M. A. Holmgren, P. E. Jönsson, and T. von Schantz. 1999. Genetic and

morphological variation in dunlin (Calidris alpina) breeding in the Palearctic tundra. Ibis 141:391-

398.

Wennerberg, L., M. Klaassen, and A. Lindström. 2002. Geographical variation and population structure in

the white-rumped sandpiper (Calidris fuscicollis) as shown by morphology, mitochondrial DNA

and carbon isotope ratios. Oecologia 131:380-390.

Wilson, W. H. 1994. Western sandpiper (Calidris mauri), The Birds of North America Online. (A. Poole,

Ed.). Cornell Lab of Ornithology, Ithaca. Retrieved from the Birds of North America Online.

Available at http://bna.birds.cornell.edu.proxy.lib.sfu.ca/bna/species/090.

http://bna.birds.cornell.edu.proxy.lib.sfu.ca/bna/species/090


8.0 STATEMENT OF LIMITATIONS

This report was prepared by Hemmera Envirochem Inc. (“Hemmera”), based on fieldwork conducted by

Hemmera, for the sole benefit and exclusive use of Port Metro Vancouver. The material in it reflects

Hemmera’s best judgment in light of the information available to it at the time of preparing this Report.

Any use that a third party makes of this Report, or any reliance on or decision made based on it, is the

responsibility of such third parties. Hemmera accepts no responsibility for damages, if any, suffered by

any third party as a result of decisions made or actions taken based on this Report.

Hemmera has performed the work as described above and made the findings and conclusions set out in

this Report in a manner consistent with the level of care and skill normally exercised by members of the

environmental science profession practicing under similar conditions at the time the work was performed.

This Report represents a reasonable review of the information available to Hemmera within the

established Scope, work schedule and budgetary constraints. The conclusions and recommendations

contained in this Report are based upon applicable legislation existing at the time the Report was drafted.

Any changes in the legislation may alter the conclusions and/or recommendations contained in the

Report. Regulatory implications discussed in this Report were based on the applicable legislation existing

at the time this Report was written.

In preparing this Report, Hemmera has relied in good faith on information provided by others as noted in

this Report, and has assumed that the information provided by those individuals is both factual and

accurate. Hemmera accepts no responsibility for any deficiency, misstatement or inaccuracy in this

Report resulting from the information provided by those individuals.

APPENDIX A

Captured Western Sandpiper Morphometrics

Port Metro Vancouver APPENDIX A Hemmera RBT2 – WESA Genetics - 1 - December 2014

Appendix A Morphometrics of Western Sandpipers Captured at Roberts Bank, Sturgeon Bank, and Boundary Bay, Delta, B.C., Spring 2012

Site Date Capture

time Band# Age

1 How aged

2 Sex3

Fat4 Wing

(mm)

Flight feather wear

5

Culmen (mm)

Tarsus (mm)

Weight (g)

DNA Analysed

Boundary Bay 19-Apr 05:30 2601-03701 ASY C + F U 3 100.0 4 24.3 26.3 25.0 -

Boundary Bay 22-Apr 07:50 2601-03702 ASY C + F M 4 98.0 4 23.1 24.8 26.0 Yes

Boundary Bay 22-Apr 07:50 2601-03703 SY C + F M 2 100.0 2 21.8 26.1 26.0 -

Boundary Bay 22-Apr 07:50 2601-03704 SY C + F F 3 103.0 2 26.6 27.1 25.5 Yes

Boundary Bay 22-Apr 07:50 2601-03705 ASY C + F M 2 96.0 4 22.2 25.3 25.0 -


Boundary Bay 22-Apr 07:50 2601-03707 ASY? C F 2 103.0 3 27.2 31.2 26.0 Yes

Boundary Bay 22-Apr 07:50 2601-03708 SY C + F M 2 92.0 2 20.4 25.3 25.0 Yes




Boundary Bay 22-Apr 08:35 2601-03712 AHY C + F M 1 94.0 3 21.5 25.2 24.0 -




Boundary Bay 24-Apr 06:10 2601-03716 ASY C + F F 2 102.0 5 25.8 26.8 26.0 Yes







Boundary Bay 24-Apr 07:15 2601-03723 SY C + F F 3 99.0 3 26.3 26.2 26.0 Yes




Site Date Capture

time Band# Age

1 How aged

2 Sex3

Fat4 Wing

(mm)

Flight feather wear

5

Culmen (mm)

Tarsus (mm)

Weight (g)

DNA Analysed


Boundary Bay 24-Apr 07:15 2601-03727 SY? C + F M 5 100.0 2 21.6 25.1 27.0 Yes

Boundary Bay 24-Apr 07:15 2601-03728 ASY C M 3 98.5 3 22.8 25.4 25.0 -


Boundary Bay 24-Apr 07:15 2601-03730 ASY C F 3 100.0 3 27.8 26.4 27.0 -

Boundary Bay 24-Apr 06:10 Not Banded - - - - - - - - -

Boundary Bay 4-May 04:30 1401-71136 ASY C + F F 4 100.0 4 26.6 26.9 28.5 -

Boundary Bay 4-May 04:30 1401-71137 ASY C + F M 4 95.0 4 22.2 25.5 25.0 -

Boundary Bay 4-May 04:30 1401-71138 SY C + F M 4 94.0 3 23.8 25.2 29.0 -

Boundary Bay 4-May 04:30 1401-71139 SY C + F M 3 95.0 3 21.4 23.1 25.0 -

Boundary Bay 4-May 04:30 1401-71140 ASY C + F M 4 98.0 3 24.0 26.2 26.0 Yes

Boundary Bay 4-May 04:30 1401-71141 SY C + F M 4 95.0 3 23.7 25.2 24.0 Yes

Boundary Bay 4-May 04:30 1401-71142 ASY C + F F 4 99.0 4 25.1 25.1 29.0 Yes

Boundary Bay 4-May 05:30 2601-03751 SY C + F F 1 100.0 2 26.1 27.1 23.0 Yes

Boundary Bay 5-May 05:00 2601-03752 SY C + F M 0 96.0 2 23.3 25.7 21.0 Yes


Boundary Bay 5-May 05:00 2601-03754 ASY C + F M 2 96.0 4 23.8 25.4 23.0 Yes




Boundary Bay 5-May 05:00 2601-03758 ASY C + F F 3 102.0 4 28.2 27.0 27.0 Yes




Boundary Bay 6-May 04:00-6:00 1401-71143 ASY C F 3 105.0 3 28.3 27.3 26.5 -

Boundary Bay 6-May 04:00-6:00 1401-71144 ASY C + F M 4 93.0 4 22.2 23.7 24.0 Yes

Boundary Bay 6-May 04:00-6:00 1401-71145 ASY C F 3 103.0 3 26.4 26.3 25.0 -


Site Date Capture

time Band# Age

1 How aged

2 Sex3

Fat4 Wing

(mm)

Flight feather wear

5

Culmen (mm)

Tarsus (mm)

Weight (g)

DNA Analysed

Boundary Bay 6-May 04:00-6:00 1401-71146 SY C + F M 2 101.0 2 24.1 26.1 24.0 Yes

Boundary Bay 6-May 04:00-6:00 1401-71147 SY C + F F 2 96.0 3 25.4 28.1 24.0 Yes

Boundary Bay 6-May 04:00-6:00 1401-71148 SY C + F M 4 97.0 1 23.1 25.8 25.5 -

Boundary Bay 6-May 04:00-6:00 1401-71149 ASY C + F F 3 103.0 4 29.5 26.8 26.5 Yes

Boundary Bay 6-May 04:00-6:00 1401-71150 ASY C + F F 4 103.0 3 26.6 26.8 28.0 -



Boundary Bay 6-May 04:00-6:00 2601-03764 ASY? C + F F 2 103.0 3 26.5 27.1 25.5 -

Boundary Bay 6-May 04:00-6:00 2601-03765 ASY C + F M 2 99.0 4 22.1 24.8 23.0 -










Boundary Bay 6-May 04:00-6:00 2601-03775 SY C + F F 3 101.0 2 26.3 26.6 25.0 -








Boundary Bay 6-May 04:00-6:00 2601-03783 ASY C + F M 3 96.0 4 22.7 24.7 22.0 Yes


Site Date Capture

time Band# Age

1 How aged

2 Sex3

Fat4 Wing

(mm)

Flight feather wear

5

Culmen (mm)

Tarsus (mm)

Weight (g)

DNA Analysed






Boundary Bay 6-May 04:00-6:00 2601-03789 ASY C + F F 4 96.0 4 25.9 25.8 27.0 Yes

Boundary Bay 6-May 04:00-6:00 2601-03790 ASY C M 2 102.0 3 23.6 26.7 27.0 -





Boundary Bay 6-May 04:00-6:00 2601-03795 AHY C + F F 4 102.0 3 27.6 27.4 28.0 -

Boundary Bay 6-May 04:00-6:00 Not Banded ASY? C + F F 3 - 3 27.2 - - -

Boundary Bay 6-May 04:00-6:00 Not Banded - - - - - - - - - -

Boundary Bay 6-May 04:00-6:00 Not Banded SY C + F M - - - - - - -

Boundary Bay 6-May 04:00-6:00 Not Banded - - - - - - - - - -

Roberts Bank 24-Apr 18:30 2601-03801 ASY C + F M 5 98.0 3 22.2 25.2 30.0 Yes


Roberts Bank 24-Apr 18:30 2601-03803 ASY C F 4 100.0 - 27.2 26.3 29.0 Yes



Roberts Bank 24-Apr 18:30 2601-03806 ASY C + F M 3 101.0 4 22.2 25.4 26.0 -


Roberts Bank 24-Apr 18:30 2601-03808 SY C + F M 3 100.0 2 22.8 26.2 27.0 Yes



Roberts Bank 24-Apr 18:30 2601-03811 SY C M 4 99.0 4 21.4 23.5 25.5 Yes


Site Date Capture

time Band# Age

1 How aged

2 Sex3

Fat4 Wing

(mm)

Flight feather wear

5

Culmen (mm)

Tarsus (mm)

Weight (g)

DNA Analysed

Roberts Bank 24-Apr 18:30 2601-03812 ASY C M 3 101.0 - 23.2 21.7 25.5 Yes


Roberts Bank 26-Apr 10:00 2601-03814 ASY C + F F 2 98.0 5 26.5 24.1 31.0 Yes

Roberts Bank 26-Apr 10:00 2601-03815 ASY C + F U 3 98.0 5 24.6 23.7 27.0 Yes


Roberts Bank 2-May 15:20 2601-03817 SY C + F M 1 91.0 2 21.9 25.4 23.0 Yes

Roberts Bank 2-May 15:20 2601-03818 SY C + F U 3 95.5 3 24.6 26.4 26.0 Yes

Roberts Bank 2-May 15:20 2601-03819 ASY C + F M 1 98.0 4 23.5 23.6 23.0 -

Roberts Bank 2-May 15:20 2601-03820 SY C + F F 3 98.0 4 26.0 26.3 32.0 -

Roberts Bank 2-May 15:20 2601-03821 ASY C + F M 2 91.0 4 22.7 26.6 26.0 Yes



Roberts Bank 2-May 15:20 2601-03824 ASY C + F F 2 100.0 4 25.4 26.8 29.0 Yes







Roberts Bank 2-May 15:20 2601-03831 SY C + F F 1 96.0 3 25.8 27.0 26.0 Yes

Roberts Bank 2-May 15:20 2601-03832 SY C + F F 1 99.0 2 34.6 24.4 25.0 Yes





Sturgeon Bank 26-Apr 05:00 1401-66045 ASY C + F F 3 103.0 4 26.7 27.4 28.5 Yes

Sturgeon Bank 26-Apr 05:00 1401-79001 ASY C + F U 2 92.0 4 24.3 27.3 21.0 -


Site Date Capture

time Band# Age

1 How aged

2 Sex3

Fat4 Wing

(mm)

Flight feather wear

5

Culmen (mm)

Tarsus (mm)

Weight (g)

DNA Analysed

Sturgeon Bank 26-Apr 05:00 1401-79002 ASY C + F M 4 96.0 - 22.9 24.7 28.5 Yes

Sturgeon Bank 26-Apr 05:00 1401-79003 ASY C + F F 4 99.0 - 27.5 27.3 27.0 -


Sturgeon Bank 26-Apr 05:00 1401-79005 ASY C + F F 3 101.0 4 26.0 26.6 30.0 -


Sturgeon Bank 26-Apr 05:00 1401-79007 ASY C + F M 4 93.0 3 22.5 24.1 25.0 Yes


Sturgeon Bank 26-Apr 05:00 1401-79009 SY C M 3 96.0 3 21.4 24.3 23.0 -




Sturgeon Bank 26-Apr 05:30 2601-03733 SY C + F M 2 96.0 3 21.7 25.8 24.0 -

Sturgeon Bank 26-Apr 05:30 2601-03734 ASY C F 3 108.0 3 27.5 27.6 28.5 -

Sturgeon Bank 26-Apr 05:30 2601-03735 ASY? C + F M 3 99.0 3 21.6 25.2 25.5 -



Sturgeon Bank 26-Apr 05:30 2601-03738 ASY C + F M 3 96.0 4 20.9 24.1 22.0 -












Site Date Capture

time Band# Age

1 How aged

2 Sex3

Fat4 Wing

(mm)

Flight feather wear

5

Culmen (mm)

Tarsus (mm)

Weight (g)

DNA Analysed

Sturgeon Bank 27-Apr 05:45 1401-71105 ASY C F 1 102.0 3 28.0 26.8 22.0 Yes

Sturgeon Bank 27-Apr 05:45 1401-71106 SY C + F M 3 93.0 2 22.7 24.3 22.0 Yes






Sturgeon Bank 27-Apr 05:45 1401-71120 ASY C F 4 101.0 3 27.0 26.1 26.0 Yes








Sturgeon Bank 27-Apr 05:45 1401-79017 ASY C + F U 2 100.0 4 24.7 27.6 25.0 -


Sturgeon Bank 2-May 04:30 1401-71112 ASY C + F F 2 97.0 5 25.5 24.6 22.5 -

Sturgeon Bank 2-May 04:30 1401-71113 ASY C + F M 3 98.0 4 23.8 26.0 25.0 Yes

Sturgeon Bank 2-May 04:30 1401-71114 SY C + F M 4 93.0 3 22.9 25.8 24.0 -

Sturgeon Bank 2-May 04:30 1401-71115 ASY C + F F 4 103.0 3 28.0 26.2 25.5 Yes


Sturgeon Bank 2-May 04:30 1401-71117 ASY C + F F 4 102.0 4 27.0 25.8 24.5 -


Sturgeon Bank 2-May 04:30 1401-71119 SY C + F M 4 96.0 2 22.9 26.0 26.5 Yes

Sturgeon Bank 2-May 04:30 1401-71122 SY C + F M 3 97.0 3 22.1 25.6 21.5 -



Site Date Capture

time Band# Age

1 How aged

2 Sex3

Fat4 Wing

(mm)

Flight feather wear

5

Culmen (mm)

Tarsus (mm)

Weight (g)

DNA Analysed





Sturgeon Bank 2-May 04:30 1401-71129 ASY C + F M 4 96.0 4 20.4 24.5 22.5 -

Sturgeon Bank 2-May 04:30 1401-71130 ASY C + F U 3 101.0 3 24.7 26.7 24.0 Yes


Sturgeon Bank 2-May 04:30 1401-71132 ASY C + F U 3 100.0 4 24.2 26.2 23.5 Yes


Sturgeon Bank 2-May 04:30 1401-71134 ASY C + F U 3 98.0 3 24.0 25.4 23.0 -


Notes: 1 Age: SY = second year (born previous year), ASY = after second year, AHY = after hatch year.

2 How Aged: C = coverts, F = feather wear.

3 Sex: M = male, F = female, U = indeterminable/unknown (culmen length: F > 24.7 mm, M < 24.2 mm).

4 Fat: 0 = none, 1 = trace covering of furculum, 2 = furculum trace to 50% full, 3 = furculum 50% to 100% full, 4 = furculum beginning to bulge, abdomen full, 5 = furculum obviously bulging, abdomen bulging, 6 = fat overflowing furculum, abdomen, in armpits, 7 = bird covered in fat.

5 Flight feather wear: 0 = heavily abraded, shafts broken, feather tips missing, 1 = all feathers tips abraded and worn; large pieces of feather tips missing, 2 = numerous nicks and wear, 3 = one of two shallow nicks in one or two feathers, 4 = minimal wear, 5 = fresh feathers, brand new, no wear.

ROBERTS BANK TERMINAL 2 TECHNICAL DATA REPORT · RBT2 – WESA Genetics - 2 - December 2014 Table 1...

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Transcript of ROBERTS BANK TERMINAL 2 TECHNICAL DATA REPORT · RBT2 – WESA Genetics - 2 - December 2014 Table 1...